Final Thesis Paper

The Effect of Dietary Nitrate, via Beetroot Juice, on High-Intensity Intermittent Exercise in

Well-Trained Male Division II Collegiate Soccer Athletes at High Altitude

By

Nicolas A. Aguila

A Thesis

Submitted in Partial Fulfillment of the

Requirements for the Degree of

Masters of Science

in Exercise Science

Department of Human Performance & Physical Education

Adams State University

2014

EFFECT OF DIETARY NITRATE IN MALE SOCCER ATHLETES 1

Table of Contents

Abstract ............................................................................................................................................6

Acknowledgements ..........................................................................................................................7

Chapter 1: Introductions...................................................................................................................8

Purpose of Study ................................................................................................................14

Research Questions ............................................................................................................14

Hypotheses .........................................................................................................................15

Delimitations ......................................................................................................................15

Limitations .........................................................................................................................16

Assumptions.......................................................................................................................16

Definition of Terms............................................................................................................16

Chapter 2: Literature Review .........................................................................................................19

Role of Nitrate, Nitrite, and Nitric Oxide in Human Physiology ......................................19

The Effect of Nitrate on Blood Pressure and Heart Rate ...................................................24

The Effect of Nitrate Supplementation on Exercise Performance .....................................25

The Effect of L-Arginine, a NO precursor, has on Exercise Performance ........................28

The Effect of Dietary Nitrate on Time-Trial Performance ................................................30

The Effect of Dietary Nitrate on Running Performance ....................................................32

The Effect of Dietary Nitrate on Intermittent Exercise ......................................................34


The Effect of Dietary Nitrate in a Hypoxic Environment..................................................35

The Effect of Dietary Nitrate on Training Status...............................................................37

Mechanisms Conferring Improved Whole Body Exercise Efficiency and Performance ..40

Reduction in the O2 Cost of Mitochondrial ATP resynthesis ............................................41

Reduction in the ATP Cost of Muscle Force Production ..................................................41

Potential Risks with Nitrate ...............................................................................................42

Summary ............................................................................................................................43

Chapter 3: Methods ........................................................................................................................44

Introduction ........................................................................................................................44

The Setting .........................................................................................................................44

The Participants..................................................................................................................45

Instrumentation ..................................................................................................................45

Research Design.................................................................................................................47

First Visit – Blood Pressure, Familiarization, and VO2max testing.....................................48

Pre-Testing .........................................................................................................................48

Supplementation and Testing Period .................................................................................49

Dietary and Training Standard ...........................................................................................50

Reliability and Validity ......................................................................................................51


Treatment of Data/Statistical Analysis ..............................................................................52

Chapter 4: Results ..........................................................................................................................54

Yo-Yo Intermittent Exercise Testing Data ........................................................................56

Resting Blood Pressure Data .............................................................................................57

Recovery Heart Rate Data..................................................................................................58

Chapter 5: Discussion ....................................................................................................................60

Recommendations ..............................................................................................................71

Chapter 6: Summary and Conclusions...........................................................................................73

Practical Applications ........................................................................................................74

References ......................................................................................................................................76

Appendix A: Research Consent Form ...........................................................................................85


Table of Figures

Figure 1. The Entero-Salivary Circulation of Nitrate in Humans..................................................21

Figure 2. The Pathway of Nitric Oxide (NO) Generation in the Human Body .............................21

Figure 3. Group mean VO2 profiles during moderate-intensity exercise across a 15 day

supplementation period with BR and PL compared with

pre-supplementation baseline ........................................................................................28

Figure 4. Pulmonary VO2 following L-arginine and PL supplementation after a

step increment to moderate exercise..............................................................................30

Figure 5. Pulmonary VO2 following L-arginine and PL supplementation after a

step increment to severe exercise ..................................................................................30

Figure 6. Diagram of Yo-Yo Intermittent Endurance Test, Level 2 (YYIETL2) .........................49

Figure 7. Individual data on the distance covered in the YYIETL2 of each participant

for each trial...................................................................................................................57

Figure 8. Individual data on resting systolic blood pressure (mmHg) of each participant

for each trial...................................................................................................................58

Figure 9. Individual data on recovery heart rates after the YYIETL2 of each participant

for each trial...................................................................................................................59


List of Tables

Table 1. Research Design...............................................................................................................47

Table 2. Baseline Testing Results ..................................................................................................54

Table 3. Beetroot Trial Testing Results .........................................................................................55

Table 4. Placebo Trial Testing Results ..........................................................................................55

Table 5. The Mean Values for all 10 participants for All Conditions ...........................................56


Abstract

Dietary nitrate has been shown to reduce the oxygen cost of submaximal exercise and improve

tolerance of high-intensity exercise, but has not been investigated in well-trained athletes at a

high altitude. Purpose: The purpose of this study was to examine the effects of chronic

ingestions of dietary nitrate via beetroot juice on high intensity intermittent exercise

performance, resting blood pressure, and recovery heart rate in well-trained male collegiate

Division II soccer athletes at an altitude of 7544 ft (2300 m). Methods: Ten well-trained male

Division II soccer players (VO2max = 57.86 ± 3.3 ml · kg-1 · min-1) were assigned in a double-

blind, randomized, crossover design to consume 140 ml of concentrated nitrate-rich beetroot

juice or placebo juice chronically, for eight days, three hours prior to exercise and preceding the

completion of a Yo-Yo intermittent endurance test, level two. Resting blood pressure was taken

prior to each test and five minute recovery heart rate was recorded after each test. Results: There

were no significant differences in the Yo-Yo intermittent endurance test, level two, between

beetroot and placebo treatments or in the recovery heart rate condition. Systolic blood pressure

showed significant decreases between the beetroot and placebo treatments and between the

beetroot and baseline treatments. Conclusions: Eight day chronic ingestion of dietary nitrate via

beetroot juice may not represent an effective strategy for enhancing high intensity intermittent

exercise in well-trained male Division II athletes at an altitude of 7544 ft (2300 m). However,

dietary nitrate may benefit hypertensive individuals and those with cardiovascular disease by

means of helping to decrease systolic blood pressure.


Acknowledgements

I would like to acknowledge and thank Dr. Tracey Robinson for her guidance and

mentoring over the last two years while I was enrolled in the HPPE graduate school at Adams

State University. I would not be here today without the countless number of hours she took to

read over my drafts and help with corrections and guide me through the whole process.

I would also like to take this time to thank my other two committee members, Dr. “Beez”

Lea Ann Schell, and Megan C. Nelson, who offered more guidance and instruction with my

thesis. Megan C. Nelson was responsible for the distribution and separation of the supplements

and placebo drink used in the study and I could not have completed my research without her.

I would like to thank the undergraduate student helpers who took hours out of their day to

help me record results and take heart rate measurements during all my trials. Thank you to

Coach Busen and the Adams State soccer team that made it possible for me to even go forth with

the study. Lastly, I would like to thank my parents, Hector (Leonardo) Aguila and Daniza

Mandich, as well as my two lovely sisters, Gabriela and Carolina, who were always there for me

whenever I needed anything and were my main support groups during my two years here at

Adams State University.


Chapter 1

INTRODUCTION

Oxygen consumption (VO2) is crucial for determining exercise capacity in human

exercise physiology. Generally, oxygen consumption increases in a linear fashion relative to

external work rate (Whipp & Wasserman, 1972). As a result, predictability in the cost of oxygen

consumption is straightforward when working at a constant rate. Therefore, if an individual can

increase his/her work rate while simultaneously decreasing oxygen utilization or maintain

current work rate while utilizing less oxygen, they could theoretically perform at a higher

workload. Oxygen consumption may be more important in conditions of low oxygen availability

(Jones, 2013). Generally, the oxygen cost of exercise is similar at low altitude as it is to sea

level; however, at higher altitudes VO2max decreases, which results in a given workload

representing a higher percentage of maximal. At any given workload, the level of exertion is

increased as the partial pressure of oxygen is reduced; therefore, when there is a decrease in

barometric pressure, oxygen consumption also decreases relative to exercise intensity, compared

to normoxia (Ibanez, Rama, Riera, Prats, & Palacios, 1993).

Oxygen consumption, for most, begins to decline at approximately 1500m with a

subsequent decline of 3% per 300m (1000ft) (Brooks, Fahey, White, & Baldwin, 2000). Recent

research, however, has shown that the cost of oxygen consumption can be decreased at

submaximal workloads by increasing dietary nitrates (Bailey et al., 2009; Bailey et al., 2010a;

Bailey et al., 2010b; Bescos et al., 2011; Ferreira, & Behnke, 2011; Jones, Bailey, & Vanhatalo,

2013; Lansley et al., 2011a; Lansley et al., 2011b; Larsen, Weitzberg, Lundberg, & Ekblom,

2007, 2010; Larsen et al., 2011; Vanhatalo et al., 2010; Vanhatalo et al., 2011). Additionally, it


has been seen that high-altitude (4200m) Tibetan residents offset physiological hypoxia and

achieve normal oxygen delivery by means of higher blood flow enabled by higher levels of

bioactive forms of nitric oxide, which is the main endothelial factor regulating blood flow and

vascular resistance (Erzurum et al., 2007). Thus, as nitrate supplementation could be beneficial

under conditions of hypoxia; its use may be more important to athletes training and competing at

high altitudes, where oxygen consumption is compromised.

By increasing the consumption of green leafy vegetables, such as spinach, lettuce,

arugula, celery and beetroot, increases in nitrate and nitrite levels occur (Bailey et al., 2009;

Bailey et al., 2010a; Lansley et al., 2011a; Lansley et al., 2011b; Vanhatalo et al., 2010). It is

recognized that dietary nitrates contain many cardiovascular benefits, such as decreasing blood

pressure in hypertensive individuals, which have the potential to increase life span (Kapil et al.,

2010; Lansley et al., 2011a; Vanhatalo et al., 2010). If a decreased blood pressure leads to

greater cardiovascular benefits, it would seem reasonable that a decreased heart rate would be

present, accompanied by a potential faster heart rate recovery. Mean arterial blood pressure is

regulated by cardiac output and total peripheral resistance (TPR) (Powers & Howley, 2012). As

previously stated dietary nitrates decrease blood pressure via vasodilatory mechanisms, i.e. TPR.

If TPR is decreased and cardiac output remains the same, blood pressure would also be reduced.

In trained individuals stroke volume is typically elevated (Powers & Howley, 2012) so it seems

reasonable that heart rate could potentially be decreased to maintain a constant cardiac output.

It has been shown that active individuals compared to sedentary individuals have lower blood

pressure, are at low risk for cardiovascular disease, and have faster heart rate recovery time post-

exercise (Anand & Jain, 2012). Additionally, active individuals will have higher maximal


oxygen uptakes, which have been correlated with improved heart rate recovery (Darr, Bassett,

Morgan, & Thomas, 1988).

Recent studies show that dietary nitrate has an effect of increasing exercise capacity

(Bailey et al., 2009; Bailey et al., 2010a; Bailey et al., 2010b; Bescos et al., 2011; Ferreira, &

Behnke, 2011; Jones et al., 2013; Lansley et al., 2011a; Lansley et al., 2011b; Larsen et al., 2007,

2010; Larsen et al., 2011; Vanhatalo et al., 2010; Vanhatalo et al., 2011). Inorganic dietary

nitrate has been reported to reduce the cost of oxygen consumption during exercise (Bailey et al.,

2009; Bailey et al., 2010b; Lansley et al., 2011a; Larsen et al., 2007, 2010) and increase

tolerance to high-intensity exercise (Bailey et al., 2009; Bailey et al., 2010a; Bailey et al., 2010b;

Larsen et al., 2010; Vanhatalo et al., 2011). Dietary nitrate is known as a possible source for

systemic generation of nitric oxide (NO) (Lundberg, & Govoni, 2004), which is important for

physiological responses to exercise such as regulating blood pressure and blood flow (Kapil et

al., 2010; Stamler, & Meissner, 2001), keeping glucose and calcium homeostatic (Stamler, &

Meissner, 2001), increasing the efficiency of oxidative phosphorylation (Clerc, Rigoulet, &

Leverve, 2007), and influencing the adenosine triphosphate (ATP) cost in muscle force

production by direct inhibition of the force-generating proteins in skeletal muscle (Galler, Hilber,

& Gobesberger, 1997). Unfortunately, the precise mechanisms that demonstrate the positive

effects that inorganic dietary nitrate supplementation have on reducing the cost of oxygen

uptake, extending time to exhaustion, or performing at a greater exercise capacity are still

unclear. Jones et al. (2013) stated that, theoretically, a lower oxygen cost during exercise at the

same power output could result from two possible mechanisms. One mechanism could be a

lower ATP cost of muscle contraction for the same force production (i.e. improved muscle

contractile efficiency) (Jones et al., 2013; Larsen et al., 2011) and/or two, a lower oxygen


consumption for the same rate of oxidative ATP resynthesis (i.e. improved mitochondrial

efficiency) (Bailey et al., 2010a; Jones et al., 2013).

Larsen et al. (2011) published research in which dietary nitrate increased plasma nitrite

and nitric oxide levels. Nitrate and nitrite have been considered stable inactive end products of

nitric oxide (Lundberg, & Govoni, 2004); however, research now suggests that there is a

different pathway that recycles nitrate and nitrite back into bioactive NO in blood and tissues

(Benjamin et al. 1994; Lundberg, & Govoni, 2004). Based on these studies it was postulated that

nitric oxide can be derived from nitrate supplementation, which could then increase oxidative

phosphorylation. Larsen et al. (2011a) showed that the simple inorganic anion, nitrate, affected

mitochondrial function as well as whole-body oxygen consumption during exercise. The authors

theorized that nitrates decrease “leakage” of protons within the inner mitochondrial membrane,

which ultimately enhance muscle mitochondria efficiency (Nair, Irving, & Lanza, 2011).

However, Bailey et al. (2010a) conducted a study suggesting that the reduced cost of oxygen

consumption is a result of the increase in muscle contractile efficiency through reduction of

skeletal muscle ATP turnover and muscle sparing the rate of PCr degradation, which in turn

reduces the total ATP cost of muscle force production.

Most recent research performed on dietary nitrate supplementation has supported the idea

that improvements occur in continuous exercise performance ranging from about six minutes to

two hours (Wylie et al., 2013b). Limited research has been completed on field sport athletes

who participate in intermittent high-intensity activities, such as soccer or lacrosse players. As

opposed to endurance and continuous activity, the stop-and-go action in intermittent high-intense

exercise utilizes a different energy demand that is not seen in a long constant work rate,


continuous exercise. As stated previously, Bailey et al. (2010a) suggested that high-intensity

exercise can be improved by sparing the rate of depletion of PCr reserves; however, the repeated

bouts of high-intensity exercise seen in sports that go from a low to high metabolic rate increases

the likelihood of fatigue in the working type II muscle fibers, which play a key role in

determining intermittent exercise performance (Colliander, Dudley, & Tesch, 1988; Krustrup et

al., 2003, 2006).

Hernandez et al. (2012) conducted a study that showed with consumption of dietary

nitrate, Ca2+ handling and contractile function of type II muscle fibers improved. Intermittent

high-intensity exercise places a lot of stress in terms of oxygen demand on the human body

which may result in the development of muscle hypoxia. These results, combined with

individuals performing in hypoxic conditions, such as altitude, place dual stress which can

potentially lead to a decrease in performance. However, recent research suggests that dietary

nitrate is particularly effective in enhancing performance in hypoxia and ischaemia (Kenjale et

al., 2011; Muggeridge et al., 2013; Vanhatalo et al., 2011). Performing at a work rate of over

50% VO2 maximum in hypoxia compared to normoxia accelerates depletion of muscle PCr and

glycogen and increases accumulation of fatigue-related metabolites (ADP, Pi, H+), which all

impair tolerance of exercise (Hogan, Richardson, & Haseler, 1999). Given these data

accumulated, it is safe to say that by implementing a dietary nitrate supplement, performance of

high-intensity intermittent exercise, such as a soccer match, at altitude, may be improved.

A majority of the research completed, looking at the influence dietary nitrate

supplementation has on continuous exercise performance, utilized valid and reliable tests;

therefore, it is just as crucial that when testing for high- intense intermittent exercise that a valid


and reliable test is incorporated. Popular protocols for testing fitness in repeated sprinting bouts

include the Yo-Yo intermittent recovery tests (IR1 and IR2) and the Yo-Yo intermittent

endurance test, level two (Yo-Yo IE2) (Bangsbo, Iaia, & Krustrup, 2008). Both Yo-Yo

intermittent tests were created to work the aerobic and anaerobic energy systems (Krustrup et al.,

2003), assess athlete’s fatigue resistance, and were specifically designed for soccer players and

soccer match play to mimic the high- intense running bouts and energy demands (Bangsbo et al.,

2008). The Yo-Yo endurance test, level two, however, has not been tested in conjunction with

dietary nitrate supplementation. The Yo-Yo intermittent endurance test, level two also

demonstrates to be a reproducible test that closely relates to running performance of soccer

players in competitive matches (Bradley et al., 2012b). Bradley et al. (2011) saw that both sub-

maximal and maximal versions of the Yo-Yo endurance test level two are highly reproducible

and are optimal for evaluating the ability to perform repeated intense exercise because the

aerobic system is heavily stimulated. Only one study to date has looked into the effects of

dietary nitrate on high- intense intermittent exercise; the researchers utilized the Yo-Yo

intermittent recovery, level one (IR1) test (Wylie et al., 2013b), which was completed at sea

level and only looked at acute effects of dietary nitrate supplementation. Bangsbo (1994)

showed that soccer players fit the ideal profile for the energy demands utilized in intermittent

sport activity. About 90% of a soccer player’s energy comes from aerobic production; however,

the anaerobic energy system is heavily taxed and plays an essential role during soccer play

(Bangsbo, 1994). Therefore, with limited research in this section of the field it would be

appropriate to see how a chronic dietary nitrate supplementation protocol, compared to acute

effects that were seen in Wylie et al. (2013b), would affect soccer player’s performance at

altitude in the Yo-Yo intermittent endurance test, level test two. A chronic protocol at altitude


may further increase the potential to perform by increasing nitrate and nitrite levels in the body

(Vanhatalo et al., 2010).

Purpose of the Study

The purpose of this study was to determine whether an eight day dietary nitrate

supplementation protocol, via concentrated beetroot juice, could increase the distance completed

in the Yo-Yo intermittent endurance test, level two at altitude in well-trained male Division II

collegiate soccer players.

Research Questions

RQ 1: Does a chronic eight day dietary nitrate supplementation protocol, via concentrated

beetroot juice, increase the distance a well-trained male Division II collegiate soccer player can

complete during an intermittent high- intensity endurance test at 7544 ft (2300 m), compared to a

placebo?

RQ 2: Does a chronic eight day dietary nitrate supplementation protocol, via concentrated

beetroot juice, reduce resting blood pressure in well-trained male Division II collegiate soccer

players at 7544 ft (2300 m)?

RQ3: Does a chronic eight day dietary nitrate supplementation protocol, via concentrated

beetroot juice, improve recovery heart rate in well-trained male Division II collegiate soccer

players during an intermittent high-intensity endurance test at 7544 ft (2300 m), compared to a

placebo?


Hypotheses

The first hypothesis of the study was that the concentrated beetroot juice would increase

the performance distance in the Yo-Yo intermittent endurance test two in the soccer players,

compared to soccer players receiving a placebo. The second hypothesis of the study was that the

concentrated beetroot juice would decrease resting blood pressure. The third hypothesis of the

study was that the concentrated beetroot juice would improve heart rate recovery.

Delimitations

The study involved a few delimitations. The research was conducted on an indoor turf

field for best simulation of a soccer match on grass. Conducting the research on outdoor grass

fields has the potential to produce more variability and could have skewed the results, such as

weather patterns. Second, the placebo supplement was a home-made placebo from black currant

berries (under .01 mmol of nitrate) comparable to the provided beetroot juice placebo from the

James White Drinks Ltd. (under .01 mmol of nitrate), but much less expensive. Thirdly, the

research was conducted solely at an altitude of 7544 ft (2300 m) and no comparisons were made

to normoxia. The study was limited to males who are engaged in the Division II collegiate

soccer program and did not include any recreational, sedentary, or female soccer players. Lastly,

the study was limited to a 140 ml of concentrated beetroot. One hundred and forty ml of

concentrated beetroot is equivalent to approximately 1 L of normal beetroot juice, which is

equivalent to about 10-11 mmol of dietary nitrate.


Limitations

Throughout each exercise performance of the Yo-Yo intermittent test, level two the

participants were encouraged to do the very best they can; however, the effort solely relied on

each individual and their motivation to complete it. Secondly, the study did not include blood

plasma concentration, which is a variable measured in most studies. Thirdly, the last day of

testing was the only day when all the participants were able to be in attendance. Lastly, the

soccer coach showed up sporadically and caused some intimidation within the participants

during trial one.

Assumptions

It was assumed that the population used in the study would respond strongly to the

dietary nitrate supplementation. The population that was utilized in the study included a group

of well-trained Division II soccer athletes who have had plenty of experience in the field and

were familiar with the test being incorporated. It was assumed that the participants would follow

the protocol and take the supplement as directed, as well as participate and finish all testing at

max effort. It was assumed that the participants believed they were ingesting two commercially

available products with no knowledge of how it would affect them. Lastly, it was assumed that

the participants could not tell the difference from the two liquids.

Definition of Terms

Adenosine Triphosphate (ATP) – used for energy metabolism and used to store energy in the

form of high-energy phosphate bonds.


High-Intensity Intermittent Exercise – repeated bouts of sprinting with little rest as seen in team-

sport play (i.e. rugby, soccer, lacrosse, football, etc.).

Hypoxia (environmental) – low partial pressure of oxygen in ambient air.

Ischaemia – an inadequate supply of blood to an organ or body part, as from an obstructed blood

flow.

Normoxia – when the partial pressure of oxygen in inspired gas is equal to the air of sea level.

Oxygen Consumption (VO2) – measure of the volume of oxygen used in the body to convert

energy into energy molecules (ATP).

Phosphocreatine – an organic compound found in muscle tissue which is capable of storing and

providing energy for muscle contraction.

P/O (Oxidative Phosphorylation) – this ratio is a measure of oxidative phosphorylation; refers to

the amount of ATP produced per pair of electrons traveling through the electron transport chain

(ETC).

Type II muscle fiber – fast-twitch muscle fibers that are recruited during high-intensity exercises,

such as repeated sprinting bouts.

Well-Trained – according to the ACSM manual, a male athlete, ages 20-29, with a VO2max of

over 56 ml · kg-1 · min-1 is considered to have superior aerobic fitness (ACSM, 2010). In the

current study, the researcher defines well-trained as those who have VO2max’s of over 56 ml · kg-

1 · min-1, while any value over 60 ml · kg-1 · min-1 represents athletes who are in an elite class.


Yo-Yo intermittent endurance test, level 2 – variation of the beep test that starts at a higher

running speed and has different increments in speed, in relation to the Yo-Yo intermittent

recovery tests and Yo-Yo intermittent endurance test, level one (Bangsbo, Iaia, & Krustrup,

2008).


Chapter 2

REVIEW OF LITERATURE

Role of Nitrate, Nitrite and Nitric Oxide in Human Physiology

In the late 1980s, the discovery of nitric oxide (NO) as an endogenous mediator of

several functions in the cardiovascular and immune system brought to light the evidence that

nitrite and nitrate are endogenously generated (Wink & Paolocci, 2008). NO plays a role in

regulating functions such as immune defense, neurotransmission, energy metabolism and other

processes (Lundberg, Weitzberg, & Gladwin, 2008). Until recently, it was previously assumed

that NO was only produced endogenously by nitric oxide synthases (NOS) from the oxidation of

the amino acid L-arginine. However, Benjamin et al. (1994) showed that nitrite derived from

dietary nitrate was a substrate for NOS-independent production of NO in the acidic conditions of

the human stomach, while Lundberg and Govoni (2004) demonstrated that nitrate can function as

a substrate for further generation of bioactive NO. Therefore, nitrite reduction to NO represents

an alternative pathway for the generation of NO which complements the NOS-derived

production (Lundberg et al., 2008).

Interestingly, ingesting dark green leafy vegetables, such as spinach and beetroot, which

have high concentrations of nitrate, increase concentrations of nitrate and nitrite in the body

(Bailey et al., 2009; Bailey et al., 2010a; Lansley et al., 2011a; Lansley et al., 2011b; Vanhatalo

et al., 2010) and also possess cardioprotective blood pressure-lowering effects (Hobbs, Kaffa,

George, Methven, & Lovegrove, 2012). Dietary nitrate enters the entero-salivary circulation

where it is then absorbed and extracted by the salivary gland and concentrated in the saliva. In


the mouth, commensal anaerobic bacteria reduces nitrate to nitrite by utilizing nitrate as an

electron acceptor during respiration (Lundberg et al., 2004). The nitrite- inducing bacteria in the

mouth are crucial for the bioavailability of nitrite because human cells are not capable of

metabolizing nitrate (Lundberg et al., 2008). Interestingly, Webb et al. (2008) showed that by

interrupting the entero-salivary circulation by spitting out all saliva the rise in plasma nitrite was

inhibited and the spitting action cancelled the potential effects of dietary nitrate. Research has

also shown that using antibacterial mouthwash eliminates the nitrate reducing bacteria in the

mouth (Govoni, Jansson, Weitzberg, & Lundberg, 2008), which in turn ablate the effects dietary

nitrate possess. When the commensal anaerobic bacteria aren’t interrupted, the converted nitrite

in the mouth is then swallowed, and two fates can occur: 1) the acidic environment of the

stomach reduces the nitrite to NO, or 2) it reenters the circulation as nitrite (Lundberg et al.,

2004) (Refer to Fig. 1 and Fig. 2 below). The mechanism of converting nitrite to NO is

expedited in hypoxia and acidosis, which are present in exercise (Lundberg et al., 2008;

Vanhatalo et al., 2010), thereby ensuring NO production in situations for which the oxygen-

dependent NOS enzyme activities are compromised. Nitrite reduction to NO and NO-modified

proteins during physiological and psychological hypoxia appear to contribute to physiological

hypoxic signaling (Lundberg et al., 2008). Thus, NO production becomes preserved in hypoxia

when NOS activity is limited to the decrease in pO2.


Figure 1 – The entero-salivary circulation of nitrate in humans (Webb et al., 2008)

Figure 2 – The pathway of nitric oxide (NO) generation in the human body. The right picture depicts

the classic L-arginine pathway and on the left the nitrate-nitrite-NO pathway (Bailey et al., 2012).


Supplementation via dietary nitrate has been a hot topic in research within the past couple

years due to nitrate’s ability to be recycled in vivo to form vasodilatory NO. Sixty to eighty

percent of person’s daily intake of nitrate comes from vegetables (Lundberg et al., 2004).

Beetroot juice, celery, lettuce, arugula, and spinach are a few listed vegetables that contain the

highest concentration of nitrate; however juice sources represent a more potent form of dietary

nitrate intake. Seventy ml of concentrated nitrate-rich beetroot juice (BR) is equivalent to 200-

250 g (about 4-5 mmol of dietary nitrate) serving of beetroot (Lundberg & Govoni, 2004), which

is significantly higher than the Acceptable Daily Intake (ADI) of 3.7 mg/kg/day (Lundberg et al.,

2008). However, the impact of extremely high concentrations of nitrate ingestion in the form of

vegetables is not considered to negatively impact health (Lundberg et al., 2004). Contrarily,

inorganic dietary nitrate in vegetable form has been shown to have various health benefits

(Bailey et al., 2012; Lundberg et al., 2004; Webb et al., 2008).

Kapil et al. (2010) concluded that dietary nitrate in the form of inorganic nitrate ingestion

provides therapeutic advantages to cardiovascular disease (CVD), such as reducing blood

pressure (BP) and increasing blood flow. Decreases in both systolic and diastolic blood pressure

readings were shown (Kapil et al., 2010). Similarly, Hobbs and colleagues (2012) demonstrated

that consumption of BR reduced systolic and diastolic blood pressure over a 24 hr period in

normotensive individuals. The research reported that inorganic nitrate via capsules and BR

increased plasma nitrite levels which consequently increased NO concentratio n. These findings

confirm that bioactive nitrite, after being reduced to NO, is important in expanding blood vessels

to increase blood flow. The positive effects that inorganic dietary nitrate showed in the above

studies could be reason why a diet high in vegetables potentially increase life-span and reduce

incidence of CVD.


Galler et al. (1997) used skinned muscle fibers from both slow- and fast-twitch rat leg

muscles to identify the mechanism of NO action in muscle contraction. The authors speculated

that NO affects the steady-state isometric tension, kinetic properties and ATPase activity. The

researchers demonstrated that NO donors producing physiologically relevant free NO

concentrations were capable of producing inhibitory effects on the mechanical properties and

ATPase activity of myofibrils (Galler et al., 1997). By inhibiting myosin ATPase, the rate of

actin-myosin attachment/detachment alters. Galler and colleagues (1997) suggested that the

effects observed in their research can be explained by a change in rate of cross-bridge cycling.

Perkins, Han, and, Sieck (1997) saw similar results with their study in rabbits: exposure of

permeabilized fibers to the NO donor SNP inhibited isometric force, Ca2+ sensitivity, and

actomyosin ATPase activity. Both of the above studies suggest that NO has a regulatory effect

on the ATP cost of force generation by altering the rate of cross-bridge cycling.

Lastly, NO has been shown to increase oxidative phosphorylation efficiency (Brown,

1995; Clerc et al., 2007). In 1995, Guy C. Brown theorized that NO reversibly inhibited

mitochondrial respiration by competing with oxygen at cytochrome oxidase. It was proposed

that NO exerted some of its main physiological and pathological effects on cell functions by

inhibiting cytochrome oxidase. Clerc et al. (2007) looked into the effect NO had on oxidative

phosphorylation efficiency by using liver mitochondria from rats to measure mitochondrial

oxygen consumption and the rate of ATP synthesis. They showed that NO binds to cytochrome

c oxidase, acting as an inhibitor of mitochondrial respiration and restricts maximal ATP

synthesis capacity, while decreasing energy waste. When there is a decrease in ATP synthesis,

all the oxygen is consumed in the generation of the proton gradient, and the ATP/Oxygen ratio is

equal to zero (Clerc et al., 2007). However, when ATP is synthesizing, part of the oxygen


consumed remains dedicated to processes unrelated to ATP synthesis, but its proportion in the

total oxygen consumption decreases when ATP synthesis increases (Clerc et al., 2007). Clerc

and colleagues (2007) showed that there was no convergence in the relationship between ATP

synthesis and oxygen consumption in the presence of NO when ATP synthesis and respiration

increased. The research concluded that NO plays a significant role in increasing oxidative

phosphorylation efficiency by reducing the leakage of the proton pump in the electron transport

chain to produce more ATP.

The Effect of Nitrate on Blood Pressure and Heart Rate

A majority of recent research has already shown that supplementation with BR or sodium

nitrate reduces resting blood pressure (BP) in normotensive and recreationally active males

(Bailey et al., 2009; Hobbs et al., 2012; Kapil et al., 2010; Lansley et al., 2011a; Larsen et al.,

2007; Wylie et al., 2013a). Systolic BP, compared to diastolic BP, seems to be the most

significantly affected measurement with nitrate supplementation, being reduced by 5-9 mmHg in

most studies. Diastolic BP and mean arterial pressure (MAP) were only affected in a few studies

(Bailey et al., 2010a; Kapil et al., 2010). A recent meta-analysis published on the effect of

inorganic nitrate and beetroot juice on BP showed that consumption was associated with greater

changes in systolic BP (- 4.4 mmHg, p < 0.001) than diastolic BP (- 1.1 mmHg, p < 0.06);

however the authors state that these effects need to be tested long-term and in individuals who

are at greater cardiovascular risk of CVD (Siervo, Lara, Ogbonmwan, & Mathers, 2013).

In addition to the effects dietary nitrate has on blood pressure, there have been limited

studies measuring the effects of dietary nitrates on heart rate, especially heart rate recovery.

Most of the studies that have looked at dietary nitrate and heart rate showed no significant


difference between the supplement and the placebo; however, those measures were exercising

heart rate and not recovery (Eliot, Heuertz, and Weiss, 2012; Larsen et al., 2007). There is

speculation that if dietary nitrates decrease BP via total peripheral resistance (Ferreira & Behnke,

2011), and cardiac output stays the same, the individual will potentially have a stronger, more

efficient cardiovascular system that has more peripheral nervous system activity, resulting in a

reduced recovery heart rate.

Effects of Nitrate Supplementation on Exercise Performance

Oxygen consumption is crucial for determining exercise capacity in human exercise

physiology. At the onset of moderate-intensity exercise, oxygen consumption increases

exponentially to reach steady state, which takes approximately 2-3 minutes in trained individuals

(Bailey et al., 2010a). In steady state, the rate of ATP breakdown is equal with the rate of ATP

resynthesis through oxidative phosphorylation. In general, oxygen uptake increases linearly

when plotted against work rate in moderate-intensity exercise (Bailey et al., 2010a). During

high-intensity exercise, the cost of oxygen consumption is elevated when carbon dioxide

production exceeds oxygen extraction (Bailey et al., 2009). Additionally, oxygen consumption

in the functioning muscle increases dramatically by means of increases in muscle blood flow

(Lansley et al., 2011a). Thus, by decreasing oxygen consumption in moderate exercise there is

potential that exercise efficiency can increase.

In 2007, Larsen and colleagues published the first study that suggested a relationship

between nitrate ingestion and changes in exercise metabolism. In their research, nine healthy,

well-trained (VO2peak = 55 ± 3.7 ml · kg-1 · min-1) males took 0.1 mmol sodium nitrate per kg of

body weight a day dissolved in water or an equal amount of sodium chloride as the placebo for


three days. Each participant went through five submaximal levels of testing followed by a time

to exhaustion test at a work rate corresponding to their calculated maximal oxygen uptake.

During this phase VO2peak was calculated. The main result from this study was that the 3 day

supplementary period of sodium nitrate decreased systolic (8 mmHg) and diastolic BP (6

mmHg), increased plasma nitrite by about 80%, and reduced the O2 cost of sub-maximal cycle

exercise by 3-4%. Absolute VO2 was, on average, 0.16 L · min-1 lower over the four

submaximal work rates prescribed. These results were surprising because the O2 cost of exercise

at given sub-maximal outputs are highly predictable. It is expected that pulmonary O2 uptake

increases by about 10 ml O2 · min-1 · W-1 of external power output during cycle ergometry.

These findings were supported by Bailey et al. (2009) in which nitrate was administered in the

form of BR. Eight healthy men (VO2max = 49 ± 5 ml · kg-1 · min-1) ingested either 0.5 L/day of

BR supplement (5.5 mmol/day) or low calorie black currant juice (PL) for six days. The

researchers revealed that hemoglobin without oxygen (deoxy-hemoglobin) amplitude was

reduced 13% after BR ingestion which indicates increased muscle oxygen delivery at the same -

O2 uptake and reduced fractional oxygen. Oxygenated hemoglobin increased at baseline during

moderate-intensity exercise, but there was no change at baseline during severe-intensity exercise.

Plasma nitrite levels were increased by 96% with BR ingestion compared to the PL and systolic

BP was decreased by 6 mmHg across all six of the sample points. The elevated levels of nitrate

and nitrite in the BR supplement group showed significant reductions in the O2 cost of cycling at

a fixed submaximal work rate (19% reduction) as well as an increased time to failure during

severe exercise (PL 583 ± 145 s vs. BR 675 ± 203 s; p < 0.05). The researchers speculated that

the amplitude of the VO2 slow-component, which is a slowly developing increase in VO2 during

constant work rate exercise performed above lactate threshold, was reduced so that exercise


tolerance increased. Also, increased muscle oxygen delivery at a constant VO2 would result in a

reduced fractional oxygen extraction in muscle. Thus, collectively, the results from both Larsen

et al. (2007) and Bailey et al. (2009) suggest that short-term, natural dietary intervention

improved efficiency of muscular work.

Larsen et al. (2007) and Bailey et al. (2009) both utilized a chronic nitrate

supplementation protocol that lasted 3-6 days; however, similar reductions in steady state VO2

during moderate-intensity cycle ergometry have been reported following acute nitrate

supplementation by Vanhatalo et al. (2010), who also tested the supplement chronically. Eight

healthy participants (including 3 females) ingested either 0.5 L /day of BR (about 5.2 mmol) or

low caloric blackcurrant juice cordial as “PL” just 2.5 hr prior to testing on day 1 (acute) and

then continued supplementation for 15 days (chronic) with repeated testing on day 5 and day 15.

The testing procedure included two 5 min bouts of moderate - intensity cycling and a ramp

incremental test to exhaustion, with all tests being separated by 10 - min recovery. The main

finding in this study demonstrated that acute supplementation of nitrate via BR, just 2.5 hr prior

to exercise on day 1, reduced steady state VO2 (about 4%) compared to PL; an effect that was

maintained when supplementation was continued for up to 15 days. Plasma nitrite concentration

(baseline: 454 ± 81 nM) was significantly elevated (+ 39% at 2.5 hr post - ingestion; + 25% at

day 5; + 46% at 15 days; p < 0.05) and systolic and diastolic BP were reduced 4% throughout

BR supplementation period. It is unclear why day 5 plasma nitrite levels weren’t elevated more

than post 2.5 hr ingestion; however, the main findings supported the idea that dietary nitrate

intake has distinct acute and chronic effects on the physiological responses to exercise (refer to

Fig. 3 below). More research is needed.


Figure 3 – Group mean VO2 profiles during moderate-intensity exercise across a 15 day supplementation period

with BR and PL compared with pre-supplementation baseline (•). The open symbols (○) show the BR supplemented

trials in A-C and PL supplemented trials in D-F (Vanhatalo et al., 2010)

The Effect L-Arginine, a NO precursor, has on Performance

Bailey et al. (2010b) investigated a related research question from the 2009 study. In this

study the authors focused on different sources of NO derivation. The study used L-arginine as a

precursor of NO and demonstrated that acute L-arginine supplementation (6 g of L-arginine

product in 500 ml of water) reduced the oxygen cost of moderate-intensity exercise and

enhanced high- intensity exercise tolerance. For 3 consecutive days, nine healthy, recreationally

active men consumed either the L-arginine supplement or the black currant cordial “PL” and

performed moderate- and severe-intensity exercises on each supplemental day, 1 hour post -


ingestion. The exercise protocol included a series of step tests of both moderate- and severe-

intensity and an incremental ramp test. The authors looked at VO2 amplitude as the difference

between baseline oxygen consumption rate and terminal exercise VO2. As a result of this

experiment, VO2 amplitude in moderate-intensity exercise was reduced by 10%, relative to the

PL. Consequently, functional gain, which is the ratio of increase in oxygen uptake per minute to

the increase in external work rate, was decreased from 10.8 ml · min-1 · W-1 in PL to 9.7 ml ·

min-1 · W-1 following L-arginine supplementation. In addition, the absolute VO2 over the final

30s of moderate-intensity exercise decreased from 1.59 ± 0.13 L/min in the PL to 1.48 ± 0.12

L/min following L-arginine supplementation and a decrease in oxygen deficit (0.45 ± 0.15 L/min

and 0.39 ± 0.12 L/min for PL and L-arginine, respectively).

In contrast to the effects seen in moderate-intensity exercise, the VO2 amplitude

increased during severe-intensity exercise, but the amplitude of VO2 slow component was

smaller, which resulted in a 20% increase in exercise tolerance via time to exhaustion (Bailey et

al., 2010b). Based on the study results, the authors concluded that the reduced cost of oxygen

consumption was due to the reduced ATP cost of force production, oxygen cost of ATP

production, or both. L-arginine and other means of nitrate supplementation (i.e. BR)

demonstrate a means to spare the utilization of anaerobic reserves such as creatine phosphate and

the accumulation of metabolites such as ADP and inorganic phosphate which related to the

fatigue process and led to improvements in exercise tolerance. Bailey et al. (2010b) concluded

that acute dietary L-arginine increased NO availability, reduced the steady-state and oxygen

consumption during moderate exercise as well as VO2 slow component, and increased exercise

tolerance in severe-intensity exercise (refer to Fig. 4 and Fig. 5 below).


Figure 4 – Pulmonary VO2 following L-arginine and PL Figure 5 - Pulmonary VO2 following L-arginine and PL

supplementation after a step increment to moderate exercise supplementation after a step increment to severe exercise.

Top: VO2 response of a representative individual data Top: VO2 response of a representative individual

Bottom: group mean VO2 response, with SD bars every 30s data. Bottom: group mean VO2 response to 6 min severe

(Bailey et al., 2010b). intensity exercise (Bailey et al., 2010b).

Effect of Dietary Nitrate on Time-Trial Performance

This chapter so far has only covered exercise that focuses on time-to-exhaustion.

However, time-to-exhaustion protocols test “exercise capacity” rather than performance per se

and are not very practical for sport due to the fact that there are no sports where the goal is to

perform for as long as possible (Curry, & Jeukendrup, 2008). According to Hopkins, Hawley,

and Burke (1999), a 15-20% improvement in time-to-exhaustion tests of exercise tolerance

correspond to a 1-2% improvement in time trial performance. In a follow-up study, Lansley et

al. (2011a) were the first authors to publish research on the acute effects of dietary nitrate

supplementation on cycling time trial performance. Nine competitive male cyclists (VO2peak =


55 ± 5.7 ml · kg-1 · min-1) ingested either 0.5 L of BR (about 6.2 mmol of nitrate) or nitrate-

depleted BR (approximately 0.0047 mmol of nitrate) as PL 2.75 h prior to either a 4- or 16.1-km

time trial (TT). The acute effects of BR ingestion showed a 2.8% reduction in 4-km TT

performance and 2.7% reduction in 16.1-km TT performance. Additionally, BR consumption

increased power output by 7-11% for the same amount of oxygen uptake. The results suggest

that dietary nitrate supplementation has the potential to benefit athletic performance in events

lasting at least 5-30 minutes in duration. The authors theorized that the increase in power output

occurred through nitric oxide-mediated improvement in muscle contractile efficiency by

reducing total ATP turnover and muscle metabolic perturbation, and subsequent reduction of

ATP cost on actin-myosin interaction or Ca2+ handling. Furthermore, nitrate supplementation

ameliorated blood flow and attenuated local blood flow-to-VO2 heterogeneities, which likely

contributed to the increase in high-intensity exercise performance. The authors concluded that

by increasing nitric oxide production via BR, improvements in power output occur without a

change in oxygen consumption so that the power output at any given VO2 is increased (Lansley

et al., 2011a).

Lansley and colleagues’ (2011a) results were supported by Cermak, Gibala, and van

Loon (2012). Twelve well-trained cyclist and triathletes (VO2peak = 58 ± 2 ml · kg-1 · min-1)

ingested 140 ml/day of concentrated BR (about 8 mmol nitrate) or nitrate-depleted BR as PL for

6 days. On day 6 of supplementing, the participants went through a 60-minute sub-maximal test

followed by a 10-km time trial on the bike with a computerized flat course in front of them. The

time to complete the computerized time-trial course was 1.2% lower with BR supplementation

and was associated with an enhanced mean power output by 2.1%. The authors also showed that

dietary nitrate supplementation via BR reduced the cost of oxygen consumption in trained


cyclists in sub-maximal efforts by 3.5% and 5.1% at work rates corresponding to 45% and 65%

Wmax, respectively. These results support the notion that nitrate supplementation improves

skeletal-muscle mitochondrial efficiency in trained cyclists.

The Effect of Dietary Nitrate on Running Performance

Most of the research completed on dietary nitrate supplementation has been done

exclusively on cyclists. Murphy, Eliot, Heuertz and Weiss (2012), however, looked into the

acute effects dietary nitrate ingestion, via whole beetroot, had on running performance. The

study incorporated five males and six females aged 18 to 55 years who had no history of

cardiovascular disease. Unlike previously cited studies, the authors created their own beetroot

juice by obtaining whole beetroots and cranberries (PL) from their local supermarket. Each

concoction was divided into 200 g portions (500 mg and 10 mmol dietary nitrate), containing an

additional 15 ml of lemon and 2 ml of ground cinnamon and nutmeg for flavoring. Following 75

minutes post-ingestion of either the BR or the cranberry placebo, the participants completed a 5

km time-trial on the treadmill. All runners were refrained from viewing running speed and time

but were allowed to adjust pace as freely as possible with the goal to complete the 5-km distance

as fast as possible. The results showed that nitrate supplementation increased treadmill velocity

by 3% (0.4 km/hr), translating to a 41 second faster finishing time. In addition, there was a 0.6

km/hr increase in running velocity the last 1.1 miles of the time-trial with consumption of BR,

compared to the placebo. The authors speculated that the late increase in running velocity of the

time-trial was due to the increasing rise in serum nitrate and concluded that consumption of 200

g of BR about 60 minutes prior to exercise enhances running performance.


Lansley and colleagues, in 2011(b), looked into the effects that dietary nitrate had on the

oxygen cost of walking and running. Nine healthy males (VO2max = 55 ± 7 ml · kg-1 · min-1) took

part in the study. For six days the participants ingested either 0.5 L/day of organic BR

(approximately 6.2 mmol) or organic nitrate-depleted BR containing approximately 0.0034

mmol of nitrate as PL. On days four and five the participants went through a treadmill exercise

test while on day six they concluded with a knee-extension exercise in order to examine muscle

phosphocreatine levels and recovery kinetics. The authors observed a reduction in the amplitude

of the pulmonary VO2 response by about 4% (PL: 1.37 ± 0.19 L/min and BR: 1.32 ± 0.23 L/min,

p < 0.05) and in the O2 cost of running 1000m by 6% (PL: 244 ± 16 ml · kg-1 · km-1 and BR: 229

± 17 ml · kg-1 · km-1, p < 0.01). Additionally, supplementation via BR showed a decrease in

oxygen consumption by 12% during baseline walking as well as a decreased VO2 value over the

last 30 s of moderate running (PL: 2.26 ± 0.27 L/min and BR: 2.10 ± 0.28 L/min, p < 0.01).

They also demonstrated a 7% reduction in oxygen consumption during severe-intensity exercise

(PL: 3.77 ± 0.57 L/min and BR: 3.50 ± 0.65 L/min, p < 0.01), which improved the exercise time-

to-exhaustion by 15% (PL: 7.6 ± 1.5 min and BR: 8.7 ± 1.8 min, p < 0.01) in all nine

participants. Muscle metabolites concentration was not significantly different with BR compared

to PL following knee-extension exercise. An elevated NO bioavailability via BR has the

potential to increase mitochondrial biogenesis through activation of cGMP-mediated pathway

(Clementi, & Nisoli, 2005); however, Lansley et al. (2011b) failed to support this hypothesis by

not seeing a difference in muscle oxidative capacity. They speculated that reduced oxygen cost

was due to a reduction in ATP cost during muscle force generation. Subsequently, the short-term

nitrate supplementation was related to the nitrite or NO-mediated effects on muscle contractile

function, rather than mitochondrial volume change (Lansley et al., 2011b).


The Effects of Dietary Nitrate on Intermittent Exercise

Most research to date that looks at the effects of dietary nitrate on performance has

primarily focused on continuous, endurance events and neglects team sport players in which

continuous sprinting bouts with little rest is the main component. Wylie and collaborators

(2013b) were the first group of individuals that looked into the effects dietary nitrate

supplementation had on team sport-specific intense intermittent exercise performance. In a

double-blind, cross-over design, fourteen recreationally active males (VO2max = 52 ± 7 ml · kg-1 ·

min-1) received either 70 ml of concentrated nitrate rich BR (4.1 mmol of nitrate) or nitrate-

depleted BR (0.04 mmol of nitrate) as PL 2.5 hr prior to exercise. One day prior to each

experimental trial the participants consumed 280 ml of BR (2x70 ml in the morning and 2x70 ml

in the evening), and on experimental days the participants consumed 140 ml 2.5 hr prior to

exercise and an additional 70 ml 1.5 hr prior. The intermittent exercise activity protocol

included the Yo-Yo intermittent recovery test, level one (Yo-Yo IR1) which is a test consisting

of repeated 20 m running bouts at a progressively increased speed interspersed by a 10 s active

recovery period. The main results of this research demonstrated that dietary nitrate increased the

distance covered in the Yo-Yo IR1 test by 4.2% compared to the PL group (BR: 1704 ± 304 m

and 1636 ± 288 m), there was an increase in blood glucose in the PL (4.2 ± 1.1 mM) compared

to BR (3.8 ± 0.8 mM), and finally, plasma [K+] was decreased in the BR group compared to PL

(p < 0.05). The authors concluded that BR has an ergogenic effect on intermittent high-intensity

exercise performance in recreational team sport players and suggest that changes in muscle

glucose uptake and muscle excitability contributed to the increase resistance to fatigue in the Yo-

Yo IR1.


Effect of Dietary Nitrate in a Hypoxic Environment

Limited research has been completed with dietary nitrate and its effect in an altitude

environment. A study completed by Muggeridge and colleagues (2013) at the University of the

West of Scotland revealed that dietary nitrate via beetroot juice can greatly benefit athletes

performing at altitude. Nine competitive male cyclists (VO2peak = 51.9 ± 5.8 ml · kg-1 · min-1)

completed three performance trials in an altitude chamber, set to a simulated altitude of 2500m,

over a three-week period. The purpose of the research was to see if beetroot juice could enhance

performance in cyclists at a high altitude, since many major cycling events take place at higher

altitudes. Each trial consisted of 15 minutes of cycling at a moderate intensity before attempting

a 16.1 km all-out time trial. The first trial was to establish a baseline so no supplements were

ingested by any of the participants. Trials two and three were both supplemented with either 70

ml of concentrated BR or PL of nitrate-depleted BR with negligible nitrate content 3 hr prior to

exercise. The results showed that plasma nitrite (PL: 289.8 ± 27.9 nM, BR: 678.1 ± 103.5 nM)

and plasma nitrate (PL: 39.1 ± 3.5 µM, BR: 150.5 ± 9.3 µM) were significantly higher in the BR

compared to PL, immediately before exercise and oxygen consumption during steady-state

exercise was lower in the BR trial (2541 ± 114 ml · min-1) than the PL trial (2727 ± 85 ml ·

min-1). However, the biggest finding from the study was that TT performance in the BR group

was significantly faster (1664 ± 14 s) than the PL group (1702 ± 15 s, p = 0.021). The authors

concluded that ingestion of BR may be a practical and effective ergogenic aid for long enduring

exercise in a hypoxic environment (Muggeridge et al., 2013)

In 2011, Vanhatalo and colleagues (2011) researched the effects dietary nitrate had in

hypoxic environments. In general, reduced atmospheric O2 availability (hypoxia) impairs


muscle oxidative energy production and exercise tolerance (Vanhatalo et al., 2011). The

researchers utilized nine healthy, moderately trained individuals (7 males and 2 females).

During the 24 hr preceding each testing protocol the participants consumed 0.75 L of nitrate-rich

beetroot juice (9.3 mmol of nitrate) or 0.75 L of nitrate-depleted beetroot juice (0.006 mmol of

nitrate) in three equal doses (0.25 L each) approximately 24 hr, 12 hr, and 2.5 hr prior. The

exercise test started with each participant breathing in normoxic or hypoxic (14.5% O2) air for 15

minutes. Shortly after the 15 minutes period of breathing the participants resumed with 4

minutes of low-intensity exercise knee extensions and, following 6 minutes of passive rest, two

24 s bouts of high- intensity exercise which were separated by 4 min of rest. The participants

then received 6 min of rest followed by a limit of tolerance (Tlim) test where they were

encouraged to go as long as possible. The researchers found that the Tlim was reduced in the

hypoxic PL group (393 ± 169 s) compared to the hypoxic BR group (471 ± 200 s, p < 0.05) and

normoxia control group (477 ± 200 s, p < 0.05). The results showed that the Tlim was not

different between the hypoxic BR group and the control (normoxia). The researchers also

showed that the overall rates of PCr degradation, Pi accumulation and pH reduction during the

exhaustive exercise bouts were greater in the hypoxic PL group than in the hypoxic BR and

control groups (all p < 0.05). Vanhatalo and colleagues (2011) demonstrated in their study that

dietary nitrate supplementation reduced metabolic perturbation during high-intensity exercise in

hypoxia and restored exercise tolerance to that observed in normoxia. The researchers also saw

that supplementation of dietary nitrate abolished the reduction in the rate of PCr recovery in

hypoxia, which the authors state could possibly be due to better NO-mediated matching of tissue

O2 supply to local metabolic rate. The research showed that nitrate supplementation has the

potential to attain the same maximal oxidative rate under extreme hypoxia as was possible in


normoxia. These findings have implication for the development of dietary interventions to

alleviate the deleterious effects of systemic hypoxia on skeletal muscle energetic and exercise

tolerance (Vanhatalo et al., 2011).

Training Status and the Effects of Dietary Nitrates

Recent evidence shows that more highly trained individuals are less likely to positively

respond to nitrate supplementation. All the studies cited previously have utilized participants

which attained VO2peak of under 60 ml · kg-1 · min-1 and were participants who were

recreationally active as opposed to ones who train for competition or train competitively for a

University or Club team. Studies completed on well-trained participants have reported no

changes in the P/O:VO2 ratio (Bescos et al., 2012; Boorsma, Whitefield, & Spriet, 2014;

Peacock et al., 2012) or a diminished enhancement in performance gains (less than or equal to

5%) (Bescos et al., 2011) compared to the results of moderately trained participants (11%)

(Larsen et al., 2011). However, training status is a topic that would benefit from more research

to result in any type of conclusion. Wilkerson et al. (2012) administered a 50 mile time trial for

eight well-trained cyclists with a VO2max of 63 ± 8 ml · kg-1 · min-1. The researcher saw that

with post ingestion of dietary nitrate in the form of BR (6.2 mmol of nitrate) there were three

non-responders and five responders (i.e. participants who’s plasma nitrite increased by greater

than 30%) with respect to BR. Of the non-responders, two did not improve time-trial

performance, whereas five of the responders reduced mean completion time by approximately

2.0%. These findings suggest a lack of change in plasma nitrite at the start of exercise may

preclude individuals from enhanced performance. Bescos and colleagues (2012) stated that a

lack of response in plasma nitrite may result due to individual differences in commensal nitrate


reductase bacteria in the mouth, or other factors that haven’t yet been uncovered. The time it

takes for plasma nitrite to peak ranges from 130-360 minutes (Wylie et al., 2013b); therefore,

individual variability for nitrite to peak could account for the lack of change in plasma nitrite

resulting in lack of enhancement in exercise performance. If individual variability in plasma

nitrite levels to peak hold true, this implies the change would be independent of training status

because it is highly unlikely that there is a difference in oral bacteria between trained and un-

trained individuals (Bescos et al., 2012). Additionally, individual variability in the time for

plasma nitrite levels to peak cannot completely explain the lack of effect in well-trained

participants, as seen in participants who display a greater than 50% increase in plasma nitrite

have failed to show improvement in performance (Bescos et al., 2012). However, Bescos et al.

(2012) speculated that the results demonstrated could be due to the fact that the low responders

were triathletes who were in the pool and swimming regularly. The presence of disinfectants

such as chlorine in the water could have interfered with the oral bacteria in a similar manner

shown with antibacterial mouthwash (Bescos et al., 2012)

Peacock et al. (2012) examined running performance in ten elite-cross country skiers

(VO2max = 69.6 ± 5.1 ml · kg-1 · min-1). This was the first study done on highly trained endurance

athletes. The participants supplemented with either 1 g of potassium nitrate (9.9 mmol of nitrate)

or a placebo capsule containing 1 g of maltodextrin 2.5 hr prior to testing, which included two 5

min submaximal running tests on the treadmill followed by a 5 km running time-trial on an

indoor track. The results showed no significant difference in mean 5 km time-trial performance

(nitrate: 1005 ± 53 s and placebo: 996 ± 49 s, p = 0.12). There were also no significant

differences in performance splits (250 meters) throughout the run with overall pacing strategy

being consistent between trials. The authors suggested that the positive effects seen in dietary


nitrate on high-intensity endurance performance may be offset in those who are well-trained

(Peacock et al, 2012). However, Peacock and colleagues (2012) did state that even though their

cross-country skiing population was experienced in producing maximal endurance running

performances, they are not running specialists. Running involves a smaller muscle mass

compared to cross country skiing, which results in lower VO2max values, which could have

influenced the results (Peacock et al., 2012), however, this research requires further investigation

Lastly, a more recent study completed by Boorsma, Whitefield, and Spriet (2014) showed

that beetroot juice does not help increase 1500 m running performance in highly trained distance

runners (VO2max = 80 ± 5 ml · kg-1 · min-1). The researchers utilized eight male 1500m runners

and randomized them in an eight day double-blind, cross-over design fashion separated by one

week. This research utilized acute (one day) and chronic (eight day) testing. The participants

ingested 210 ml of concentrated BR (19.5 mmol nitrate) or placebo (PL) each day and completed

a submaximal treadmill run and 1500 m time trial on an indoor 200 m track after day one and

day eight. The results showed that plasma nitrate increased from 37 ± 15 to 615 ± 151 µm

(acute) and 810 ± 259 µm (chronic) following BR consumption. However, even though the

results showed an increase in plasma nitrate levels, no VO2 difference were seen at 50, 65 and

80% VO2peak and 1500 m time trial was unaffected. The authors did note that two of the

participants did improve significantly in the 1500 m time trial (acute: 5.8 ± 5.0 s; chronic: 7.0 ±

0.5 s). The authors concluded that for the majority BR does not help improve 1500 m

performance in elite runners; however, there might be some exceptions (Boorsma, Whitefield, &

Spriet, 2014).


Mechanisms Conferring Improved Whole Body Exercise Efficiency and Performance

Several potential mechanisms have been proposed to explain the improved exercise

efficiency and performance observed following nitrate supplementation. In general, the

decreased energy cost of exercise at any given fixed power output could result from 1) a

reduction in the O2 cost of mitochondrial ATP resynthesis, 2) a reduction in the ATP utilization

of ATPases associated with muscle force production (acto-myosin ATPase, sarco-endoplasmic

Ca2+ -ATPase (SERCA) and/or Na+/K+ -ATPase), and/or 3) compensatory increase in the ATP

provision from the substrate phosphorylation (Bailey et al., 2010b). However, it is not possible

for a compensatory increase in PCr breakdown and anaerobic glycolysis to completely augment

ATP production because the magnitude of the reduction in oxygen consumption during exercise

following nitrate supplementation would far exceed the capacity for anaerobic energy

production. Furthermore, a compensatory increase in substrate level phosphorylation is not

supported in the research. Plasma lactate has consistently been shown to not increase following

nitrate supplementation compared to placebo during moderate- and high-intensity fixed work

rate exercise or time-trial cycling tests where the participants self select their power outputs

(Bailey et al., 2009; Bescos et al., 2012; Cermak et al., 2012; Larsen et al., 2007; Peacock et al.,

2012, Wilkerson et al., 2012; Wylie et al., 2013b) Additionally, muscle pH measured by P-MRS

and estimates of glycolytic ATP contribution are the same between nitrate and placebo

supplementation (Bailey et al., 2010b). Therefore, it is postulated that nitrate supplementation

improves exercise economy by improving mitochondrial respiratory efficiency and/or by

reducing the ATP cost of contraction.


Reduction in the O2 Cost of Mitochondrial ATP Resynthesis

Evidence shows that nitrate supplementation increases the ATP yield of mitochondrial

oxidative phosphorylation by decreasing proton leak across the inner mitochondrial membrane.

Larsen et al. (2011) observed an astounding 19% increase in P/O ratio (oxidative

phosphorylation) (Nitrate: 1.62 ± 0.07; Placebo: 1.36 ± 0.06, p = 0.02) during sub maximal ADP

stimulation in isolated mitochondria post nitrate supplementation. The results also showed that

post nitrate supplementation, the respiratory control ratio (RCR), which is the ratio between state

3 and state 4 respiration, was higher compared to the placebo (Nitrate: 8.5 ± 0.7; Placebo: 6.5 ±

0.7). State 3 respiration is the rate of oxygen consumption in the presence of ADP and substrate

while state 4 respiration is the rate of oxygen consumption when all ADP has been re-

phosphorylated to ATP. This outcome infers that nitrate treatment creates better coupling

between respiration and oxidative phosphorylation. Additionally, mitochondrial respiration with

substrates, but without added ADP, termed LEAK respiration, commences as compensation for

proton slippage. Post nitrate treatment LEAK respiration was reduced 45% compared to the

placebo. Therefore, it was suggested that the reduction in proton slippage or leakage occur both

during exercise and resting state (Larsen et al., 2011). These results support the notion that

nitrates decrease “wastage”, effectively increasing the amount of ATP generated per unit of

oxygen consumed (Nair, Irving, & Lanza, 2011).

Reduction in the ATP Cost of Muscle Force Production

Skeletal muscle ATP turnover during contraction is predominantly determined by the

activity of the actomyosin ATPase and the sarcoendoplasmic reticulum calcium ATPase

(SERCA), with a smaller contribution from the Na+/K+ ATPase (Bailey et al., 2010a). Bailey


and colleagues (2010a) discovered that dietary nitrate (consequently, more than doubling plasma

nitrite) reduced the degree of phosphocreatine (PCr) degradation during both low- and high-

intensity exercise, which ultimately resulted in sparing of PCr stores in the body without altering

pH levels after exercise (Bailey et al., 2010). During both intensity bouts the changes in oxygen

consumption and PCr were extremely proportional following supplementat ion. Bailey et al.

(2010a) reported that if the reduction in oxygen cost during exercise was exclusively due to an

increase in mitochondrial oxidation, then muscle PCr and ADP accumulation would not have

changed. Additionally, the researchers showed a reduction of muscle ATP turnover for a given

work rate which is thought to occur via inhibition of the actomyosin-ATPase and the Ca2+-

ATPase. Thus, the authors claim that the reduction of oxygen consumption during exercise is

principally a result of a reduced ATP turnover rate and improvements in high-intensity exercise

associated with muscle sparing the rate of PCr degradation (Bailey et al. 2010a).

Potential Risks with Nitrate

The association of nitrate and nitrite in fruits and vegetables with decreased cancer and

cardiovascular risk is overshadowed by health risks, including gastrointestinal cancer in adults,

associated with nitrite-mediated nitrosation to produce carcinogenic N-nitrosamines (Tang,

Jiang, & Bryan, 2011). However, nitrate and nitrite in vegetable form seem to differ from other

means of nitrate and nitrite ingestion. Nitrite is popularly used in processed meats and one study

conducted by Kilfoy and colleagues (2011a) showed that high nitrate and nitrite consumption

from processed meat increased the risk of pancreatic cancer non-significantly (p = 0.11), while

total inorganic dietary nitrate intake had no correlation to the cancer (Kilfoy et al., 2011a). In

another study conducted by Kilfoy et al. (2011b), the researchers found that two years and more


of intaking nitrate (88 mg/day) and nitrite (1.2 mg/day), which was determined by the National

Institutes of Health-American Association of Retired Persons (NIH-AARP) diet, resulted in an

increased risk of thyroid cancer. Even though nitrate and nitrite in processed meats was thought

to be the risk factor of common cancers, Kilfoy et al. (2011b) state that vegetable and fruit intake

reduced the risk of cancers (i.e. pancreatic and thyroid). Tang et al. (2011) state in their review

that dietary nitrate in the form of beetroot juice is extremely beneficial. Eating more vegetables

and fruits and less meat or animal products has beneficial effects on reducing the risk of some

cancers and cardiovascular disease (Tang et al., 2011). Thus, it can be suggested that by

increasing one’s consumption of fruits and vegetables an athlete may help increase performance

by decreasing their chances of cardiovascular disease.

Summary

In consideration of all the research that has been discussed, dietary nitrate has

demonstrated to lower the cost of oxygen consumption; however, its effects on intermittent high-

intensity sports are still relatively new to research. If dietary nitrate in the form of BR and dark

green leafy vegetable can enhance intermittent exercise performance, such as a soccer match,

especially those that occur in a hypoxic environment, it would seem logical to promote changes

in the diet to increase dietary nitrate levels. Therefore, the purpose of this research was to

expand our current knowledge of dietary nitrate by means of concentrated BR and investigate its

effects in male Division II collegiate soccer athletes who consistently train and play at 7544 ft

(2300 m) elevation.


Chapter 3

METHODS

Introduction

Research has shown that dietary nitrate supplementation via beetroot juice (BR) enhances

exercise performance by decreasing oxygen consumption at submaximal efforts (Bailey et al.,

2009; Bailey et al., 2010a; Bailey et al., 2010b; Bescos et al., 2011; Ferreira & Behnke, 2011;

Jones et al., 2013; Lansley et al., 2011a; Lansley et al., 2011b; Larsen et al., 2007, 2010; Larsen

et al., 2011; Vanhatalo et al., 2010; Vanhatalo et al., 2011). However, there are limited data

supporting the case that chronic ingestion of inorganic dietary nitrate is an effective supplement

for improving high- intensity intermittent performance and even more limited studies have been

done in hypoxic conditions. Only one study to date has looked into the effects of a chronic BR

supplemental period lasting longer than six days (Vanhatalo et al., 2010). Vanhatalo and

colleagues’ (2010) chronic study lasted up to 15 days of supplementation. In the current study,

further research was performed to see how ingestion of chronic dietary nitrate would affect

intermittent exercise at an elevation of 7544 ft (2300 m). The study was done by utilizing

NCAA Division II male collegiate soccer athletes. In order to examine this relationship, an eight

day cross-over, double-blind, placebo controlled experiment was used, separated by a six-day

washout period.

The Setting

The experiment was conducted indoors on artificial turf in the Adams State University

athletic training facility, specifically, in the Adams State University Indoor Track and Field


“Bubble”. Completion of the Yo-Yo intermittent endurance test, level two, took place on turf

surfacing as opposed to a synthetic track.

Participants

Ten well-trained male soccer players from the Adams State University Soccer team

(mean ± SD: age 19.4 ± 0.09 years, height 1.78 ± .04 m, body mass 73.2 ± 8.5 kg, VO2max =

57.86 ± 3.3 ml · kg-1 · min-1) familiar with intense intermittent exercise volunteered to participate

in this study. All of the participants had at least four years of competitive soccer experience. All

testing was completed during the participant’s off-season. The participants were individuals free

of tobacco use and other dietary supplements. The participants were instructed to refrain from

using antibacterial mouthwash and chewing gum during the supplementation period.

Additionally, the participants gave their written informed consent (Appendix A) to participate

after the experimental procedures, associated risks, and potential benefits of participation had

been explained in detail. The study was approved by the Adams State University Institutional

Review Board.

Instrumentation

There are several instruments used in the study:

CD player: CD of Yo-Yo intermittent endurance test level two was played in a CD player with

loud enough speakers for the participants to hear.

Cones: Cones were marked out on the turf field 20 m apart so the athletes know when to turn

around.


Heart rate monitor: Heart rates of all participants were recorded throughout the maximal

treadmill test and throughout the intermittent exercise test. Recovery heart rates were taken.

Metabolic Cart: The ParvoMedics TrueOne Metabolic System (OUSW 4.3.3) assessed

pulmonary gas exchange and O2 consumption throughout the incremental cycle ergometer test.

Motorized Treadmill: The treadmill was used to assess each participant’s VO2max.

Placebo: The placebo was in the form of concentrated black currant cordial. The juice was

diluted to match the consistency of the concentrated beetroot and had an added hint of lemon for

flavor. The placebo had negligible nitrate content. The blackcurrant cordial was made and

placed in the exact same bottles as the beetroot by a neutral third party who is affiliated with the

Adams State HPPE department.

Sphygmomanometer and Stethoscope: Two instruments used to manually attain blood pressure

readings.

Supplement: The dietary nitrate supplement was in the form of 2x70 ml (140 ml total)

concentrated beetroot juice. This liquid is equivocal to approximately 1.0 L of beetroot juice.

Each 2x70 ml container contained 10-11 mmol of nitrate (600-800 mg) and was ingested 2.5-3

hours prior to each day’s exercise training and testing days. A higher concentration of nitrate

was used compared to the recommended amount (0.5 L of beetroot juice/4.1-5.0 mmol or about

400 mg nitrate) because the participant pool were well-trained athletes that might need more

nitrate to show any effects. The appearance, consistency and texture of the concentrated beetroot

matched the placebo as close as possible; however, some noticeable differences did not matter

due to the fact that all participants completed both conditions and didn’t know the experimental


hypothesis until completion of the study. The beetroot, provided by James White Drinks,

Ipswich, UK, came in their standard 70 ml bottle. James White Drinks provided the researcher,

at a subsidized cost, with empty 70 ml bottles for the placebo (black currant juice cordial) to be

placed in. The empty bottles were the exact replicas of the actual beetroot. The placebo liquid

was placed in the empty bottles by a third party and were each labeled with a letter to determine

supplement or placebo (i.e. subject #1A = suppl., #1B = placebo).

Turf Field: The Adams State University athletic facility (The Bubble) was utilized, which is

made of artificial turf. There will be plenty of space for the participants to complete testing

without obstructions.

Timer: The timing device was instructed on an iPhone using the application “bleep test”.

Research Design

The experiment used a double-blind, placebo controlled cross-over design, as seen in Table 1.

Table 1

The Layout of the Research Design Schedule

Week 1 Monday Tuesday Wednesday Thursday Friday Saturday Sunday

VO2 VO2 VO2 VO2 VO2 ---- Pre-test


S/P S/P S/P S/P S/P S/P S/P

Week 3

Monday Tuesday Wednesday Thursday Friday Saturday Sunday

S/P +

YYIE2 Wash Wash Wash Wash Wash Wash



Week 5

Monday Tuesday Wednesday Thursday Friday Saturday Sunday

S/P +

YYIE2 ---- ---- ---- ---- ---- ----

Note. VO2 = VO2max testing and initial resting BP readings; Pre-test = YYIE2 pre-test S/P = Supplement/Placebo

Period; YYIE2 = Testing Day; --- = nothing complete; Wash = Washout period


First Visit – Blood Pressure, Familiarization, and VO2max Testing

The participants reported to the Human Performance Laboratory on four separate

occasions over a five week period for each experimental trial. During the first visit to the

laboratory each participant’s resting blood pressure readings were taken, each participant was

familiarized with the Yo-Yo intermittent endurance test, level two (Yo-Yo IE2), and performed

an incremental treadmill exercise test (Precor USA 956i) using a metabolic cart (ParvoMedics

TrueOne Metabolic System-OUSW 4.3.3) to determine VO2max (ACSM, 2010). The protocol for

the treadmill test had stages that lasted two minutes in duration and began at 0% gradient. The

first stage the participants started at 3 mph, second stage at 6 mph, and then each subsequent

stage after increased by 1 mph until volitional exhaustion. If the participant did not reach

volitional exhaustion by stage six, 10 mph, then only the gradient was increased at a 2% gradient

with no increase in speed, again, until volitional fatigue. After the completion of the incremental

test, the participants were randomly assigned in a crossover design and received eight days of

dietary nitrate supplementation with either nitrate (NO3-; 10-11 mmol/day; administered as 2x70

ml concentrated organic BR/day; Beet It Sport, James White Drinks, Ipswich, UK) or “placebo”

(PL; low-calorie concentrated blackcurrant cordial with negligible nitrate content). The PL,

low-calorie concentrated blackcurrant cordial, included the following ingredients: blackcurrant

berries, water, and a hint of lemon for taste. Following the eight day supplemental period the

participants completed the high-intense intermittent exercise test (Yo-Yo IE2).

Pre-Testing

Following the VO2max testing, anywhere from 3-6 days, later in the week, on Sunday, the

participants arrived to the “Bubble” and completed the pre-testing for the Yo-Yo IE2. The Yo-


Yo IE2 tests, as seen in Figure 6, were performed on an indoor turf field at a length of 20 m to

simulate indoor soccer matches. Prior to testing, the participants engaged in a 10 minute warm-

up at a self selected pace. The maximal version of the Yo-Yo IE2 test consisted of repeated

2x20 m shuttle runs, marked off by two cones, at progressively increasing speeds dictated by an

audio bleep emitted from a CD player. Between each shuttle the participants had a five second

active recovery jogging period, circling a cone 2.5 m behind the finish line. When a participant

twice failed to reach the finishing line in time, the distance covered, when done, was recorded in

meters and was representative of the test result.

Heart rate (HR) was recorded continuously throughout the experiment by a Polar Fitness

heart rate monitor. HR upon completion of the test was recorded. Five minutes after the

participant completed the test, recovery HR was recorded (Pierpont & Voth, 2004). It was

speculated that a potential decrease in blood pressure through dietary nitrates may also help with

an athlete’s recovery heart rate time after exercise, as stated in the previous chapters.

Supplementation and Testing Period

Participants arrived at the lab three hours prior to each practice and/or trial and ingested

2x70 ml of concentrated BR or placebo so the researcher could observe appropriate ingestion and

2.5 meters 20 meters

Figure 6 – Diagram of the Yo-Yo intermittent endurance test, level 2 (YYIETL2)


ensure adherence to research protocol. On the testing days (Yo-Yo IE2), the participants were

free to continue with their daily normal activities after ingestion of the BR or PL, but were asked

to refrain from any strenuous activity, such as running or playing any high-intensity intermittent

sport. Participants then arrived to the “Bubble” two and a half hours later where resting BP was

be recorded, a 5 minute familiarization of the Yo-Yo IE2 test was run, followed by 20 minutes of

passive rest. This served to assess the reproducibility of the physiological responses to repeated

intermittent exercise. Heart rate (HR) was recorded continuously throughout the experiment and

recovery HR was assessed five minutes post-testing.

A six day wash-out period separated each supplemental period to ensure that the

participants were back to baseline values (Larsen et al., 2007; Vanhatalo et al., 2010) and had

adequate recovery. The order between the nitrate and PL supplementation periods were

randomized and balanced. Prior to any testing, the participants were unaware of the

experimental hypothesis and were informed that the purpose of the study was to compare

physiological responses in exercise following the consumption of two commercially available

products. The personnel administering the exercise test was not aware of the type of beverage

being consumed by the participants. An individual within the HPPE department was responsible

for labeling and handling distribution.

Dietary and Training Standardization

All participants were instructed to refrain from using antibacterial mouthwash and

chewing gum during the supplementation period because these products have been shown to

eliminate the commensal anaerobic bacteria in the mouth required to convert nitrate to nitrite

(Govoni et al., 2008). All participants were also asked to refrain from spitting after ingestion of


the PL or BR because this process has been shown to interrupt the enterosalivary circulation and

block the rise in plasma nitrite concentration (Webb et al., 2008). The participants were not

asked to refrain from eating foods naturally high in nitrate, such as spinach and arugula, so that

the study reflects the most accurate, natural application of BR supplementation. Prior to each

exercise test, the participants were advised to eat and drink as they normally would when

preparing for a soccer match. Each participant kept a 24 hr food record preceding the testing

trials so they had the ability to replicate the exact methodology for each subsequent test. All of

the participants were asked to stay hydrated leading up to each trial and instructed to refrain from

strenuous activity, caffeine and alcohol 24 hr preceding testing sessions. Each individual

maintained similar levels of activity (volume and intensity) and exercise training eight days

preceding the trials while following the supplement regimen. Exercise testing was performed at

the same time of day (8:30 pm), each testing day (Monday) with the exception of baseline

testing, which was performed at 2:00 pm on a Sunday (as opposed to Monday).

Reliability and Validity

In order to assess intermittent high- intensity sport activity it is crucial that there is a test

that highly resembles the energy systems of match play. Only one study to date has looked at the

effects that dietary nitrate supplementation has on intermittent sport exercise performance and

they utilized what’s called the Yo-Yo intermittent recovery test, level 1 (Yo-Yo IR1). This type

of test assesses an athlete’s fatigue resistance and taxes both aerobic and anaerobic system while

has being extremely reproducible in team sport players (Krustrup et al., 2003). However, the

present research study utilized the Yo-Yo intermittent endurance test, level two (Yo-Yo IE2),

which varies differently from the Yo-Yo IR1. Bradley, Mascio, Bangsbo and Krustrup (2012a)


recently demonstrated a strong correlation between the Yo-Yo IE2 test performance and high-

intensity running (r = 0.67, p < 0.01) using a large number of elite players. The same research

showed a correlation between Yo-Yo IE2 performance and change in high-intensity running

during match-play through various stages of the season (Bradley et al., 2011). Bradley et al.

(2011) demonstrated Yo-Yo IE2 reproducibility by comparing their result with other intermittent

high-intensity exercise tests. The test-retest coefficient of variability (CV) of 3.9% that Bradley

and colleagues (2011) achieved was very similar to values obtained for the Yo-Yo IR1 as well as

values obtained from Bradley et al. (2012b) who tested the Yo-Yo IE2 on elite female soccer

athletes. Consequently, heart rate values obtained post testing during the sub-maximal version

also exhibited high reproducibility, with a CV value of 1.4% (Bradley et al., 2011). In addition,

Bradley et al. (2012b) showed that the Yo-Yo IE2 is extremely valid by observing a large

correlation between Yo-Yo IE2 test performance vs. total distance (r = 0.55, p < 0.05) and high-

intensity running (r = 0.70, p < 0.01).

In conclusion, the Yo-Yo IE2 test has been shown to be highly reproducible and is a valid

assessment tool that can be used as an indicator of match-specific physical capacity in soccer

players. Additionally, the Yo-Yo IE2 performance test illustrates high sensitivity by

differentiating between performances of players in various stages of the season, playing position

and age groups.

Treatment of Data/Statistical Analysis

Repeated measures ANOVA was used to analyze the data in SPSS software, version 21.

The independent variables were BR and PL conditions, and dependent variables were resting BP,

recovery HR, and the distance covered in the Yo-Yo IE2 test. Post-hoc tests, via Bonferonni


were used to determine where any statistical differences lie. Statistical significance was accepted

at p < 0.05 and results are presented as a mean ± SD unless stated otherwise.


Chapter 4

RESULTS

A total of 10 male participants (mean ± SD: age 19.4 ± 0.09 years, height 1.78 ± .04 m,

body mass 73.2 ± 8.5 kg, VO2max = 57.86 ± 3.3 ml · kg-1 · min-1) participated in this study. The

individual results for each condition are displayed below in Table 2, Table 3, and Table 4. The

mean values for all 10 participants and each condition are displayed below in Table 5. The

average VO2max of the selected participants was 57.86 ± 3.3 ml · kg-1 · min-1. These results

indicate that the participants who were selected were well-trained and were in superior shape

(ACSM, 2010).

Table 2

Baseline Testing Results

Baseline Testing

Subject VO2max (ml · kg-1 · min-1) Distance (m) BP (mmHg) HRmax(bpm) HRpost(bpm)

1 50.9 840 119/70 162 103

2 54.9 1480 118/58 177 107

3 61.5 1740 118/72 196 118

4 56.4 --- 120/62 --- ---

5 60.7 1620 115/68 184 127

6 57.4 1700 120/68 189 126

7 57.4 1600 120/70 181 105

8 60.2 2120 115/64 211 139

9 61.4 1800 120/62 197 122

10 57.8 1840 126/58 196 114


Table 3

Beetroot Trial Testing Results

Subject Distance (m) BP (mmHg) HRmax(bpm) HRpost(bpm)

1 940 136/78 167 108

2 1060 124/68 173 101

3 2120 120/68 187 115

4 1720 128/78 172 112

5 1480 116/72 190 124

6 1700 104/62 192 127

7 2160 108/62 178 99

8 1900 108/58 213 138

9 1960 108/50 188 165

10 --- 116/68 --- ---

Table 4

Placebo Trial Testing Results

Subject Distance (m) BP (mmHg) HRmax(bpm) HRpost(bpm)

1 640 122/68 177 111

2 1600 124/68 171 113

3 1600 110/64 191 107

4 1240 130/68 187 121

5 1640 106/62 183 109

6 2400 110/70 189 113

7 1640 118/72 180 124

8 2520 102/58 212 131

9 1680 122/70 195 115

10 2400 122/72 193 108


Table 5

The Mean Values for all 10 participants for All Conditions.

Baseline Testing

Subjects VO2max (ml ·

kg-1 · min-1) Distance (m) SBP (mmHg) DBP (mmHg) HRmax(bpm) HRpost (bpm)

Means of

10

participants

57.86 ± 3.3

1623.5 ± 349.5 119.3 ± 3.2 66.0 ± 4.9 188 ± 14 117.9 ± 11.9

Beetroot

Distance (m) SBP (mmHg) DBP (mmHg) HRmax(bpm) HRpost (bpm)

1657.5 ± 346.5 112.3 ± 7.0* 66.0 ± 4.9 184 ± 14 121 ± 20.7

Placebo

Distance (m) SBP (mmHg) DBP (mmHg) HRmax(bpm) HRpost (bpm)

1736 ± 577.6 118.0 ± 5.2 68.2 ± 4.4 189 ± 11 115.2 ± 7.8

Note. There was a significant difference and decrease in resting systolic BP between the baseline testing and

beetroot testing (p = .03) as well as the placebo testing and beetroot testing (p = .03). (*) marks the significance at

the p < 0.05 level. No significant difference was seen between the baseline testing and the placebo testing.

Yo-Yo Intermittent Exercise Testing Data

The results showed that there were no significant differences between the trials (baseline,

beetroot, and placebo trials), F(2, 18) = 0.68, p = 0.52, d = .04, and the researcher accepts the

null hypothesis. The average distances completed for each trial and all ten participants were the

following: 1623.5 ± 349.5 meters for baseline, 1657.5 ± 346.5 meters for beetroot, and 1736 ±

577.6 meters for the placebo groups. Figure 7 below shows each individual participant’s data.


Figure 7 – Individual data on the distance covered in the YYIETL2 of each participant for each trial.

Resting Blood Pressure Data

The results showed that systolic blood pressure was significantly different between the

trials, F(2, 18) = 8.292, p = .003, d = 0.48. These results reject the null hypothesis and accept the

research hypothesis that dietary nitrate via beetroot juice helps decrease systolic blood pressure.

The Bonferonni post-hoc test showed that there were significant differences between baseline

(119.3 ± 3.2 mmHg) and beetroot trial (112.3 ± 7.0 mmHg) (p = .03), and the placebo (118.0 ±

5.2 mmHg) and beetroot trial (112.3 ± 7.0 mmHg) (p = .03). No significant differences were

revealed between the baseline and placebo values. Figure 8 below shows each individual

participant’s data.

0

500

1000

1500

2000

2500

3000

Part. 1 Part. 2 Part. 3 Part. 4 Part. 5 Part. 6 Part. 7 Part. 8 Part. 9 Part. 10

Dis

tan

ce C

ove

red

(m)

Participants

Baseline

Beetroot

Placebo


Figure 8 – Individual data on resting systolic blood pressure (mmHg) of each participant for each trial.

No significant differences were identified between diastolic blood pressure values F(2,

18) = .838, p = .449, d = .085. Diastolic blood pressure stayed relatively consistent with all three

trials (baseline = 66 ± 4.9 mmHg, placebo = 68.2 ± 4.4 mmHg, beetroot = 66.0 ± 4.9 mmHg).

(Refer to Table 5).

Recovery Heart Rate Data

No significant differences were found between recovery HR, F(2, 18) = .369, p = .696,

d = .039 in any of the conditions. These results mean that the researcher accepts the null

hypothesis that recovery HR was not affected by dietary nitrate via beetroot juice consumption.

Average recovery heart rates for baseline, beetroot, and placebo trials were 117.9 ± 11.9 bpm,

121 ± 20.7 bpm, and 115.2 ± 7.8 bpm, respectively. Figure 9 below shows each individual

participant’s data.

0

20

40

60

80

100

120

140

160


Systo

lic B

lood P

ressu

re (m

mH

g)

Participants

Baseline

Beetroot

Placebo


Figure 9 - Individual data on recovery heart rates after the YYIETL2 of each participant for each trial.

Note. Participant four was unable to attend his baseline trial due to undisclosed reasons. Participant 10 was unable to attend his

BR trial due to a concussion but was able to attend his baseline and placebo trials. No differences were seen in participant 10’s

baseline and placebo trial indicating that there was no placebo effect with his trial.

0

20

40

60

80

100

120

140

160

180

200


Recove

ry

Heart

Rate

(bpm

)

Participants

Baseline

Beetroot

Placebo


Chapter 5

DISCUSSION

One finding of this study was that dietary nitrate supplementation, in the form of 2x70 ml

bottles of concentrated beetroot juice (10-11 mmol of dietary nitrate), did not significantly

improve high intense intermittent exercise performance at an altitude of 7544 ft (2300 m). The

current study revealed that the distance completed in the Yo-Yo IR2 test was not improved upon

a chronic eight day supplementation protocol. This finding does not support the experimental

hypothesis.

Recently, beetroot juice has received a lot of attention from scientific research, among

athletes and coaches, and in the media, as potential for its ergogenic effect. Inorganic beetroot

contains a naturally high level of nitrate, which has been confirmed as the main functional

component responsible for its ergogenic properties (Lansley et al., 2011b). Dietary nitrate enters

the entero-salivary circulation where it is absorbed and extracted by the salivary gland and

concentrated in the saliva. In the mouth, commensal anaerobic bacteria reduces nitrate to nitrite

by utilizing nitrate as an electron acceptor during respiration (Lundberg et al., 2004). The nitrite-

inducing bacteria in the mouth are crucial for the bioavailability of nitrite because human cells

are not capable of metabolizing nitrate (Lundberg et al., 2008). This conversion process has

been reported to raise plasma nitrate and nitrite levels (within 2.5 hours) as well as been shown

to increase performance (Lansley et al., 2011a; Lansley et al., 2011b; Murphy et al., 2012).

Lansley and colleagues (2011a) showed that acutely ingesting 6.2 mmol of dietary nitrate 2.5

hours prior to exercise in the form of beetroot juice (BR) reduced 4 km time trial (TT)

performance by 2.8% and 16.1 km TT performance by 2.7%. Additionally, the authors showed


that 6.2 mmol BR consumption increased power output by 7-11% for the same amount of

oxygen uptake. The results suggest that dietary nitrate supplementation has the potential to

benefit athletic performance in events lasting at least 5-30 minutes in duration. The authors

theorized that the increase in power output occurred through nitric oxide-mediated improvement

in muscle contractile efficiency by reducing total ATP turnover and muscle metabolic

perturbation, and subsequent reduction of ATP cost on actin-myosin interaction or Ca2+ handling

(Lansley et al, 2011a). The current study, which utilized 10-11 mmol of dietary nitrate,

contradicts the results that were shown in the study completed by Lansley et al. (2011a). The

participants who completed the Yo-Yo intermittent endurance test, level two, in the current study

all averaged around the same distances for each trial and no significance difference was seen

between all three conditions (baseline: 1623.5 ± 349.5 meters; beetroot: 1657.5 ± 346.5 meters;

placebo: 1736 ± 577.6 meters). It is unclear as to why no performance effect was seen in the

current study; however, it is speculated that the presence of the soccer coach influenced the

results, which will be discussed later in the chapter.

Although, in the current study, no performance benefits were achieved with the chronic

eight day supplementation period, there was a significant decrease in systolic blood pressure,

which has been shown in other research studies (Bailey et al., 2009; Hobbs et al., 2012; Kapil et

al., 2010; Lansley et al., 2011a; Larsen et al., 2007; Wylie et al., 2013a). These results are

consistent and support the secondary hypothesis proposed within this research study. Systolic

blood pressure decreased by an average of 7 mmHg. Consistent with other studies, there was no

significant change with diastolic blood pressure (Lansley et al., 2011a; Larsen et al., 2007). The

result of a decrease in systolic blood pressure provides further evidence that ingesting dietary


nitrate in the form of beetroot juice may help individuals who are at risk for cardiovascular

disease, such as heart failure and arrhythmia (Lundberg, Carlstrom, Larsen, & Weitzberg, 2011).

Blood pressure and cardiovascular diseases are an issue in today’s U.S. society. The

results on resting blood pressure found in the current research provide further benefit as to why

beetroot and other vegetables are important for the American diet. In 2013, Bond and colleagues

looked at the effects of dietary nitrate, in the form of beetroot juice, on systemic and

cerebrovascular hemodynamics. They hypothesized that beetroot juice could decrease blood

pressure and cerebrovascular resistance (CVR). In order to accomplish this, the authors utilized

12 female subjects who were all subjected to either a control group or beetroot juice group. The

researchers demonstrated that the beetroot juice treatment group had a lower cerebrovascular

resistance index, systolic blood pressure, and heart rate-systolic pressure product. These findings

suggested improved systemic and cerebral hemodynamics that could translate into a dietary

treatment for hypertension (Bond et al., 2013). As stated previously, ingesting dark green leafy

vegetables, such as spinach and beetroot, which have high concentrations of nitrate, possess

cardioprotective blood pressure-lowering effects (Hobbs et al., 2012). In 2012, Shay and

colleagues showed results in their research that individuals who ingested higher intakes of fruits,

vegetable protein, fiber, magnesium, non-heme iron, potassium and consumed lower intakes of

processed meats, high-fat dairy, and sugar-sweetened beverages all experienced low long-term

rates of cardiovascular disease mortality and greater longevity.

The last finding in the current study concluded there were no significant differences (p =

.696) between recovery heart rates in all of the trials. This result does not support the research

hypothesis of achieving faster recovery heart rate post dietary nitrate consumption. It was


speculated that the potential for a faster heart rate recovery was high. Because blood pressure

has been shown to decrease in previous studies with the consumption of dietary nitrates (Hobbs

et al., 2012 ), as well as in the current study, it would seem reasonable that heart rate recovery

would also be enhanced since they are both related. Blood pressure is the product of cardiac

output and total peripheral resistance, and cardiac output is the product of heart rate and stroke

volume (Powers & Howley, 2012). In a trained athlete, such as those in the current study, stroke

volume is typically higher than the average sedentary individual. In order to counteract this

increase in stroke volume, the trained individual’s heart rate then begins to decrease to maintain

a similar cardiac output as before but being more efficient with each heart pump (Powers &

Howley, 2012). Speculating from this and based on known cardiovascular physiology (Powers

& Howley, 2012), if dietary nitrate decreases resting blood pressure in trained individuals via

higher blood flow circulating to the recovering muscles due to reduced total peripheral

resistance, more blood will be traveling to the heart at a faster rate. If more blood travels to the

heart at a faster rate and stroke volume is high in these trained individuals, it seems reasonable

that the heart then does not have to be pumping as hard and the individual’s cardiovascular

system is working at a more efficient rate. This efficiency would come from more peripheral

nervous system activity, thus a faster recovery heart rate. This is the first study to look at dietary

nitrates and the effect they have on recovery heart rate. As stated previously, all recovery heart

rates were similar across trials and no significant differences were shown. More research is

needed in this area to assess and support these results.

To the researcher’s knowledge this is only the second study to investigate the effects of

dietary nitrate on high- intensity intermittent activity in athletes via Yo-Yo testing, and the first

study to investigate the effects of a chronic eight day dietary nitrate supplementation on high-


intensity intermittent activity in trained athletes at high altitude (7544 ft/2300 m). It was an

interesting finding that the participants did not improve in the high intensity intermittent activity,

especially at such a high elevation. In a study done by Erzurum and colleagues (2007), the

researchers saw that Tibetan highlanders’ main mechanism of counteracting the detrimental

effects of high altitude hypoxia (4200 m) is due to their higher blood flow enabled by their

naturally higher levels of bioactive forms of nitric oxide. The authors reported that the Tibetan

highlanders had more than double the forearm blood flow of low-altitude residents, resulting in a

greater sea level oxygen delivery to tissues. Tibetans also had a more than 10-fold higher

circulating concentration of bioactive nitric oxide products, including plasma and red blood cell

nitrate (Erzurum et al., 2007). From this study it may seem reasonable that the participants in the

current study should have improved in performance; however, that was not the case. The

difference potentially lies in the differences of elevation presented in Erzurum and colleagues

(2007) (4200 m/13770 ft) and the current study (2300 m/7544 ft).

It has been reported that supplementation via beetroot may improve performance during

repeated high-intensity intervals on a rowing ergometer (Bond, Morton, & Braakhuis 2012).

Bond et al. (2012) asked rowers to complete 6 x 500 m rowing ergometer repetitions (about 90 s

completion time per repetition) with 90 s recovery. The authors identified that the dietary nitrate

supplementation group displayed a higher mean power output, which could have implications for

improving performance in high-intensity interval training sessions. Following Bond et al.’s

(2012) study, it seems that high intensity intermittent exercise, in the form of a Yo-Yo test,

would be improved with dietary nitrate supplementation, especially in a hypoxic environment.

In fact, Wylie et al. (2013) utilized the Yo-Yo intermittent recovery test level one and showed

that ingestion of 490 ml (35-38.5 mmol dietary nitrate) concentrated beetroot juice improved


performance (at sea-level). However, what differed from Wylie et al. (2013) and the present

research study was that Wylie et al.’s (2013) participants were not trained and the researchers

administered 3.5 times more concentrated beetroot (490 ml/35-38.5 mmol compared to

140ml/10-11 mmol dietary nitrate) than what was administered in the current research study.

Therefore, it may be concluded that in the current study training status had an effect in addition

to not administering enough dietary nitrate. There is a chance that the participants could not

convert the nitrate to nitrite for there to be any type of ergogenic effect and help with

performance that was already at a high level. The researcher speculates that well-trained athletes

might have a different and unknown mechanism as well as a different chemistry of enzymes in

the mouth of converting nitrate to nitrite compared to sedentary individuals. Peacock et al.

(2012) speculated this as well in their research with their elite cross-country athletes. The

conversion process is a separate part of this research that needs to be studied more thoroughly.

It has been shown in studies that training status does have an effect when it comes to

utilization in the dose of dietary nitrate (Bescos et al., 2012; Boorsma, Whitefield, & Spriet,

2014; Peacock et al., 2012). Peacock et al. (2012) saw no increase in performance when ten elite

cross-country athletes (VO2max = 69.6 ml · kg-1 · min-1) consumed dietary nitrate, acutely, 2.5

hours prior to testing. As stated previously, Wilkerson et al. (2012) administered a 50 mile time

trial for eight well-trained cyclists with a VO2max of 63 ± 8 ml · kg-1 · min-1. The researchers

saw that post-ingestion of dietary nitrate in the form of BR (6.2 mmol of nitrate) resulted in three

non-responders and five responders (i.e. participants who’s plasma nitrite increased by more than

30%) with respect to BR. Of the non-responders, two did not improve time-trial performance,

whereas five of the responders reduced mean completion time by 2.0%. This relationship

between non-responders and responders could have been the case in the current study. As can be


seen in the results with recovery heart rate there was an individual who looked to be an outlier,

which may be an indication of a non-responder in the current study. However, after completion

of the outlier labeling rule, the participant was not seen as an outlier. Recovery heart rate data

looked to be off and indicate that they might be a non-responder; however, participant nine’s Yo-

Yo results and blood pressure results seemed to suggest otherwise; participant nine had a

decrease in systolic blood pressure as well as an increase in Yo-Yo testing performance. It is

unclear as to why this particular participant had an unusually slow recovery heart rate and more

research is needed to fully understand the reasoning. When taking all measured conditions into

consideration (Yo-Yo testing, blood pressure and recovery heart rate), the researcher speculates

one of two outcomes. The first speculation is that within the current research there was only one

true responder to the beetroot juice out of the 10 participants. This individual, participant seven,

had a VO2max of 57.4 ml · kg-1 · min-1 and demonstrated an increase in the distance covered with

the high intensity intermittent Yo-Yo test, a decrease in systolic blood pressure, and lastly, a

slightly faster recovery heart rate. The second speculation is that within the current research

there was one outlier and that was participant seven who was the only individual to show

improvements in distance covered, a decrease in systolic blood pressure as well as an improved

recovery heart rate.

Wilkerson et al. (2012) findings suggest a lack of change in plasma nitrite at the start of

exercise may preclude individuals from enhanced performance. In addition to Wilkerson et al.’s

(2012) research, Bescos and colleagues (2012) stated that a lack of response in plasma nitrite

may result due to individual differences in commensal nitrate reductase bacteria in the mouth, or

other factors that have not yet been uncovered. Boorsma, Whitefield, and Spriet (2014) saw no

significant improvements in eight elite 1500 m runners (VO2max = 80 ± 5 ml · kg-1 · min-1) with


19.5 mmol dietary nitrate supplementation on 1500 m track performance. The 19.5 mmol

dietary nitrate the researchers used is approximately double the amount that was used in the

current study. Only two of the eight subjects had better 1500 m performance times.

Additionally, the researchers did see plasma nitrate levels increase acutely (37 ± 15 to 615 ± 151

µm) and even more so chronically, over eight days (870 ± 259 µm). These results suggest that

most of the athletes were responders; however, no significant improvements in athletic

performance came from the increases in plasma nitrate. It would seem reasonable that if all

subjects had a significant increase in plasma nitrate they would have all increased their

performance times, which was not the case in Boorsma, Whitefield, and Spriet’s (2014) research.

Some of the research cited above does not seem to explain the reasoning of why there

was a lack of an increased Yo-Yo performance within the current study. Most of the research

cited above that showed no significant results or increases in performance all included athletes

with a VO2max of over 60 ml · kg-1 · min-1, whereas in the present study the participants’

VO2max’s were just under 60 ml · kg-1 · min-1. The mean VO2max values for the participants in the

current study was 57.86 ± 3.3 ml · kg-1 · min-1; however, this mean VO2max result was recorded at

an elevation of 7544 ft (2300 m), while the other studies were all completed at sea-level. The

researcher speculates that there may be a certain threshold of VO2max values and anyone over that

threshold might need to ingest more dietary nitrate, via beetroot juice. In Cermak, Gibala, and

van Loon’s (2012) research, their 12 well-trained cyclist and triathletes recorded VO2peak values

of 58 ± 2 ml · kg-1 · min-1. In their study, the participants ingested similar amount of beetroot

juice (140 ml/day of concentrated BR or about 8-10 mmol of dietary nitrate) and nitrate-depleted

beetroot juice as their placebo for 6 days. These authors showed that dietary nitrate

supplementation via beetroot juice reduced the cost of oxygen consumption in trained cyclists in


sub-maximal efforts by 3.5% and 5.1% at work rates corresponding to 45% and 65% Wmax,

respectively. Based off of this study it seems reasonable to speculate that individuals who have

VO2max values over 60 ml · kg-1 · min-1 might not benefit from ingesting dietary nitrate

concentrations of 4-10 mmol. These individuals might need a higher dosage of dietary nitrate to

see any type of ergogenic effect. Since in the current study the VO2max testing results were

recorded at a high elevation (7544 ft), the researcher speculates that the mean VO2max value of

57.86 ± 3.3 ml · kg-1 · min-1 would be a higher value if the study had taken place at sea-level. If

the participants were to complete the VO2max test at sea-level the average would be higher

because the partial pressure of oxygen is increased (Powers & Howley, 2012); thus, that average

would ultimately be higher than the speculated VO2max threshold of over 60 ml · kg-1 · min-1

(Wilmore & Costill, 2005). However, the researcher also speculates that since some of the

participants were born and raised at a high elevation, the VO2max test is true regardless of

elevation. The difference between sea-level and altitude born participants was not taking into

account; therefore, future research at high elevation should consider where the athlete was born

and raised. More research is needed in this area to uncover the relationship between VO2max

values and dosages of dietary nitrate.

Since the present research study did not include any plasma nitrite or nitrate measures

from any of the participants there lies a possibility that individual variability was a factor or as

stated, the participants did not convert enough dietary nitrates to nitrite. Future research should

measure plasma nitrate and nitrite levels. Lastly, there is a possibility that the majority of the

participants in the current research were non-responders and the beetroot juice that was ingested

simply had no effect on their performance, no matter how much they would ingest. More


research is needed in this area to determine individual variability as well as the percentage of

individual variance between responders and non-responders in well-trained athletes.

The findings that dietary nitrate had no effect on performance of intermittent activity at

altitude was contradictory to Muggeridge and colleagues (2013) research who looked at the

effects of dietary nitrate, via beetroot juice, at a high altitude in well-trained cyclists. In general,

reduced atmospheric O2 availability (hypoxia) impairs muscle oxidative energy production and

exercise tolerance (Vanhatalo et al., 2011). It has been cited that the conversion pathway of

nitrate-to-nitrite is expedited in these hypoxic conditions (Vanhatalo et al., 2011). Therefore, it

seems reasonable and plausible that dietary nitrate supplementation would be extremely

beneficial in these conditions (Vanhatalo et al., 2011). In the limited research that has been done

at altitude, Muggeridge and colleagues (2013), as well as Vanhatalo and colleagues (2011),

reported significant performance improvements at a high elevation (2300 m/7546 ft) similar to

the elevation of the current study or other conditions that induce hypoxia. None of the studies

were done at high intense intermittent activity, potentially suggesting that there is a different

process that occurs with high-intensity exercise at altitude compared to continuous endurance

exercise.

Lastly, there is a possibility that the lack of performance results was due to the

participants’ coach being present during one of the trials and, in the researcher’s opinion,

skewing the participants’ mental state. The current study had a strict protocol and the first trial

that occurred post supplementation was not strictly followed. The strict protocol design was not

followed by the head soccer coach; that is to say, he would pull the participants out of the trial

when he deemed them complete. This was contrary to the researcher who administered the


testing. The participants were told to keep going because they had not violated any of the

protocol’s instruction. According to the participants, because of intimidation and fear of getting

yelled at, they listened to the head coach’s orders and ignored the strict protocol instructions of

the researcher as to when they had completed the test. Additionally, the participant pool was

limited during the first two trials. Participant four was not able to show up during the baseline

testing (trial one) for undisclosed reasons and participant ten was not able to show up for his

beetroot trial due to his health. Participant ten suffered a concussion the week prior to his

beetroot trial. The participant who missed the baseline testing was available for the placebo and

beetroot trials and the participant who missed the beetroot trial was available for baseline testing

and the placebo trial. Trial three was the only trial where all ten of the participants were present

as well as no coach.

Supplementation with dietary nitrate, in the current study, did not help improve recovery

heart rate or diastolic blood pressure but did significantly improve systolic blood pressure, which

supports the researcher’s alternative hypothesis. Foods derived from vegetables that have high

nitrate and nitrite levels have been suggested to have a “positive impact on human health, by

directly countering high blood pressure and limiting platelet aggregation and endothelial

dysfunction that set the ground for chronic vascular process such as atherosclerosis (p. 618)”

(Wink & Paolocci, 2008). The current findings in this research provide evidence that ingesting

foods with a high concentration of dietary nitrate, from vegetables, may help reduce systolic

blood pressure and potentially help hypertensive individuals but not necessarily help improve

high intensity intermittent exercise in well-trained soccer athletes at a high altitude.


Recommendations

Based on the current findings, future research is needed in this field of study. Studies

have already shown improvements in performance with acute ingestion of dietary nitrate (Bailey

et al., 2010a; Lansley et al., 2011a; Lansley et al., 2011b; Vanhatalo et al., 2010); however, it is

important to note that chronic ingestion (more than 3 days) of dietary nitrate utilizing high

intensity intermittent exercise has not been researched extensively, especially at high elevation.

This may have been the first study to investigate the effects of a chronic eight day dietary nitrate

supplementation on the Yo-Yo intermittent endurance test, level two, in Division II well-trained

soccer athletes at a high altitude. While no performance effects were identified, it is important

that a better controlled environment be utilized for future research. Additionally, having a large

participant pool and having more beetroot juice, and thus higher nitrate levels, may further

enhance the current study and be utilized for future research. The participants were ingesting

140 ml (10-11 mmol of dietary nitrate) of concentrated beetroot; however, more may have been

necessary for this particular participation group. As more research becomes published, more

information on the proper dosage will be known. The biggest factor in this research was the lack

of control with the research and the participants’ coach. It is hard to determine if an effect would

have been seen if there was not any confusion, but another study with the same protocol may

help with the issue.

Additionally, more chronic studies with dietary nitrate should be utilized with

future research. This study utilized eight days of supplementation, while Vanhatalo and

colleagues (2010) used a chronic 15 day supplementation protocol. Boorsma, Whitefield, and

Spriet (2014) utilized eight days, just like the current study, and saw a greater plasma nitrate


concentration on day eight than they did on day one with all of their participants. More doesn’t

necessarily mean better; however, the researcher speculated that utilizing a more chronic

protocol (over 15 days) has potential to benefit athletes. If more chronic studies take place then

it may be easier to make comparisons to dietary nitrate’s acute effect. It is speculated that

dietary nitrate levels may plateau at certain dosages and after a certain number of days into

supplementation (Vanhatalo et al., 2010); therefore, determining the range of this plateau and the

daily dosage will help identify more accurately the proper means of dietary nitrate

supplementation via beetroot juice. Additionally, it is speculated by the researcher that acute

ingestion might show improvements just as positive as chronic supplementation (Vanhatalo et

al., 2010); however, more research is needed in this area to find out if that is the case.

Future research should entail more diverse population groups such as females, rugby

players, power-lifters, football athletes, baseball, and more studies on endurance runners.

Utilizing various types of sports that rely on different energy systems could potentially show

what sports will benefit more from dietary nitrate, if there is any ergogenic effect at all. Funding

was a limitation in the current study so having a large enough participant pool where

supplementation of dietary nitrate could be administered with different dosages to different

groups would be a great start. Additionally, future research should entail a testing protocol that

is valid and reliable on the population of participants being used. The Yo-Yo intermittent

endurance test, level two, is an example of a reliable and valid test to assess soccer player

performance, just like a 3000 m, 5000 m, and 10000 m time trial would be a valid and reliable

test for endurance runners.


Chapter 6

SUMMARY AND CONCLUSION

The results of this study indicate that a chronic eight day supplementation of dietary

nitrate did not confer any significant benefit to well-trained Division II soccer players

performing high intensity intermittent exercise in the form of the Yo-Yo intermittent endurance

test, level two at a high altitude. Within each trial, the participants’ distance covered were

similar (1623.5 ± 349.5 meters for baseline, 1657.5 ± 346.5 meters for beetroot, and 1736 ±

577.6 meters for the placebo groups). These results did not support the researcher’s hypothesis

and accepted the null hypothesis. In addition, the results of this study indicated that a chronic

eight day supplementation period of dietary nitrate does not help accelerate recovery heart rate at

an elevation of 7544 ft (2300 m). The study did show a decreased systolic blood pressure in the

participants, which indicates that beetroot juice or other green leafy vegetables high in dietary

nitrate may help provide positive benefits in reducing risk for cardiovascular disease. By

demonstrating that beetroot juice helps decrease systolic blood pressure in well-trained athletes,

other athletes or non-athletes and those who are at risk for cardiovascular disease may use this

information and may translate it to a healthier lifestyle by altering their current dietary protocols

and eating these types of foods more often. Lastly, further studies are necessary to explore

whether dietary nitrate can enhance performance in other elite groups (i.e., endurance running

performance, lacrosse, etc.) and conditions (i.e., more altitude studies), whereas research

adopting a more basic-science and reductionist approach (i.e., measuring plasma nitrate and

nitrite levels; responders vs. non-responders) is required to explore dietary nitrates’ and nitric

oxides’ specific mechanisms.


Practical Applications

Chronic supplementation of dietary nitrate via beetroot juice was shown to not improve

athletic performance (high intensity intermittent performance) in well-trained Division II soccer

athlete at an elevation of 7544 ft (2300 m). However, most of the research shows that sedentary

or untrained individuals can benefit greatly from this type of nutritional supplementation (Bailey

et al., 2010b; Lansley et al., 2011a; Lansley et al., 2011b). By ingesting 4-5 mmol of dietary

nitrate in the form of beetroot juice 2.5 hours prior to exercise, blood pressure can be decreased

via vasodilatation, which has the potential to help with an increase in performance.

The current research found that 140 ml of concentrated beetroot juice (10-11 mmol

dietary nitrate) significantly lowered systolic blood pressure in well-trained Division II male

soccer athletes at high altitude. Because in the current study well-trained athlete were the main

participant pool, this result suggests that beetroot juice has high potential in helping individuals

with high blood pressure, individuals with other cardiovascular diseases, and individuals who are

not well-trained. By decreasing blood pressure in a hypertensive individual, they may be able to

do more work in a given day and live a healthier lifestyle. Chronic supplementation of beetroot

juice has potential to help improve health, which is big news in today’s age. By increasing the

consumption of green leafy vegetables, such as spinach, lettuce, arugula, celery and beetroot,

increases in nitrate and nitrite levels will occur (Bailey et al., 2009; Bailey et al., 2010a; Lansley

et al., 2011a; Lansley et al., 2011b; Vanhatalo et al., 2010), which help with reducing systolic

blood pressure and in turn will help with cardiovascular disease such as hypertension.

As far as high intensity intermittent exercise activity, the current study showed no

improvements in well-trained individuals; however, dietary nitrate in the form of beetroot juice


has potential to help untrained individuals. By consuming dietary nitrates (4-5 mmol up to 35-

38.5 mmol dietary nitrates) in the form of beetroot juice, blood pressure may be decreased via

vasodilatation. When this occurs in the body more blood can be transported at a faster rate to the

working muscles. More blood transportation to the working muscles means that more oxygen is

transported as well. As stated previously, maybe well-trained individuals need a higher dose to

see any effect. More research is needed in this field of study to come up with any conclusive

evidence.


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Appendix A: Research Participant Consent Form

Adams State College

Request to obtain approval for the use of human participants – expedited review

Date : November 27th, 2013

To: Brent King, ASU Institutional Review Board

Name : Nick Aguila

Email: [email protected]

Mailing Address: 912 8th street, Alamosa, CO 81101

Phone: 860-874-7193

Responsible Faculty Member

Chair of Thesis Committee: Tracey Robinson, Ph.D.

Email: [email protected]

Phone: 719-587-7663

Subject: The Effect of Dietary Nitrate via Beetroot Juice on High-Intensity Intermittent Exercise in Male

Division II Collegiate Soccer Athletes

Others in Contact with Human Participants :

Research Assistants: HPPE Assistant Visiting Professor, Megan Nelson; HPPE Graduate Assistant,

Clayton Foster; HPPE Graduate Assistant, Maria Martinez; and Graduate Athletic Trainer, Aaron Ellis

The title of the research: The Effect of Dietary Nitrate via Beetroot Juice on High-Intensity Intermittent

Exercise in Male Division II Collegiate Soccer Athletes

Objectives of the research: Beetroot juice has been suggested as a means to help improve exercise

performance. Beetroot juice, as a natural drink, has been shown to have high amounts of dietary nitrate,

which is then converted to nitric oxide (NO). NO has been shown to reduce risk of cardiovascular disease

and additionally, NO is important for physiological responses to exercise such as regulating blood

pressure and blood flow, keeping glucose and calcium homeostatic, increasing the efficiency of oxidative

phosphorylation, and influencing the adenotriphosphate (ATP) cost of muscle force production by direct

inhibition of the force-generating proteins in skeletal muscle. Additionally, consumption of beets, beet

juice and other green leafy vegetables has been shown to reduce the risk of cardiovascular disease. Thus,

the primary purpose of this research is to determine if an 8-day cross-over dietary nitrate supplementation

protocol, via beetroot juice, separated by an 6-day washout period, can increase the distance covered in

the Yo-Yo intermittent exercise test, level 2 (Yo-Yo IE2). Variables measured will include: heart rate,

blood pressure, and distance traveled in the Yo-Yo IE2.


It is hypothesized that the consumption of this readily available product, beetroot juice, will

increase the distance covered in the Yo-Yo intermittent endurance test, level 2.

Methods of procedure :

The Setting

The experiment will be conducted indoors on artificial turf in the Adams State University athletic

training facility, specifically, the field house in Plachy Hall. Completion of the Yo-Yo intermittent

endurance test, level 2, will take place on turf surfacing as opposed to a track.

Participants

The participants of the study will include members of the Adams State University men’s soccer

team, ranging from age 18 to 23 years, with a minimum of 4 years of competitive soccer experience. The

participants will perform all exercise tests in their off-season. The participants will be individuals free of

tobacco use and other dietary supplements. The participants will also be instructed to refrain from using

antibacterial mouthwash and chewing gum during the supplementation period because it has been shown

that these actions will ruin the process of converting nitrate to nitrite to NO. Out of the 34 athletes on the

roster, the cross-over study will incorporate 10 of them. The 10 chosen will be the most reliable players

that their coach knows will follow research protocol and will also choose to volunteer. This number is

used in case a couple of the participants don’t complete the protocol and their data needs to be dropped.

Research Design

Table 2 - VO2 = VO2max testing and initial resting BP readings; Pre-test = YYIE2 pre-test; S/P =

Supplement/Placebo Period; YYIE2 = Testing Day; --- = nothing complete; Wash = Washout period


VO2 VO2 VO2 VO2 VO2 ---- Pre-test




S/P + YYIE2 Wash Wash Wash Wash Wash Wash




S/P + YYIE2 ---- ---- ---- ---- ---- ----


Testing

The experiment will use a double-blind, placebo controlled cross-over design, as seen in Table 1.

The participants will report to the laboratory on 4 separate occasions over a 5 week period for each

experimental trial. During the first visit to the laboratory each participant will get resting blood pressure

readings taken, be familiarized with the Yo-Yo intermittent endurance test, level 2 (Yo-Yo IE2), and

perform an incremental treadmill exercise test (Precor USA 956i) to determine VO2 max. The protocol

for the treadmill test will have stages that last 2 minutes in duration and at 0% gradient (ACSM manual).

The first stage the participants will start at 3 mph, second stage at 6mph, and then each subsequent stage

after will increase by 1 mph until volitional exhaustion. If a participant has not reached volitional

exhaustion by stage 6, 10 mph, then only the gradient will start to increase at 2% gradient with no further

increase in speed, again, until volitional fatigue. After the completion of the incremental test, the

participants will randomly be assigned in a crossover design and receive 10 days of dietary nitrate

supplementation with either nitrate (NO3-; ~0.8g/day; administered as 2x70ml concentrated organic

BR/day; Beet It Sport, James White Drinks, Ipswich, UK) or “placebo” (PL; low-calorie concentrated

blackcurrant cordial with negligible nitrate content). The PL, low-calorie concentrated blackcurrant

cordial, will include the following ingredients: blackcurrant berries, water, and a hint of lemon for taste.

Following the 8 day supplemental period the participants will undergo the high-intense intermittent

exercise test (Yo-Yo IE2).

There is one known side effect that can occur with the consumption of beetroot juice and that is

beeturia, which turn the consumer’s urine and stool pink/purple. Beeturia occurs in 10-14% of the

population who consume beets and is a natural part of the digestion for beets.

Pre-Testing

Following the VO2 max testing, later in the week and with at least 1 day rest, the participants will

arrive at the field house to complete pre-testing for the Yo-Yo IE2. The Yo-Yo IE2 tests, as seen in the

figure below, will be performed on an indoor turf field at a length of 20m to simulate indoor soccer

matches. Prior to testing, the participants will engage in a 10 minute warm-up at a self selected pace.

The maximal version of the Yo-Yo IE2 test consists of repeated 2x20m shuttle runs, marked off by two

cones, at progressively increasing speeds dictated by an audio bleep emitted from a CD player. Between

each shuttle the participants will have a five second active recovery jogging period, circling a cone 2.5m

behind the finish line. When a participant twice fails to reach the finishing line in time, the distance

covered, when done, will be recorded in meters and will be representative of the test results.

2.5 meters 20 meters


Heart rate (HR) will be recorded continuously throughout the experiment by a Polar Fitness heart rate

monitor. Five minutes after a participant is completed with the test, recovery HR and recovery BP

readings will be taken.

Subjects will be tested on this same Yo-Yo IE2 test after the 8-day supplement trial and again after the 8-

day placebo trial.

Supplementation and Testing Period

Participants will arrive at the lab three hours prior to each practice and/or trial and ingest 2x70ml

of concentrated BR or placebo so the researcher can observe appropriate ingestion and ensure adherence

to research protocol. On the testing days (Yo-Yo IE2), the participants will be free to continue with their

daily normal activities after ingestion of the BR or PL, but will be asked to refrain from any strenuous

activity, such as running or playing any high-intensity intermittent sport. Participants will then arrive at

the field house two hours later where resting BP will be recorded, a 5 minute familiarization of the Yo-Yo

IE2 test will be run, followed by 45 minutes of passive rest. This serves to assess the reproducibility of

the physiological responses to repeated intermittent exercise. Heart rate (HR) will be recorded

continuously throughout the experiment and recovery HR and BP will be assessed five minutes post-

testing.

A 6-day wash-out period will separate each supplemental period to ensure that the participants are

back to baseline values and have adequate recovery. The order between the BR and PL supplementation

periods will be randomized and balanced. Prior to any testing, the participants will be unaware of the

experimental hypothesis and will be informed that the purpose of the study is to compare physiological

responses in exercise following the consumption of two commercially available products. The personnel

administering the exercise tests will not be aware of the type of beverage being consumed by the

participants. An individual within the HPPE department will be responsible for creating the PL and

handling distribution.

Dietary and Training Standardization

All participants will be instructed to refrain from using antibacterial mouthwash and chewing

gum during the supplementation period because these products have been shown to eliminate the

commensal anaerobic bacteria in the mouth required to convert nitrate to nitrite. All participants will also

be asked to refrain from spitting after ingestion of the PL or BR because this process has been shown to

interrupt the enterosalivary circulation and block the rise in plasma nitrite concentration. The participants

will be asked to maintain their current diet so that the study reflects the most accurate application of BR

supplementation. Prior to each exercise test, the participants will be advised to eat and drink as they

normally would when preparing for a soccer match. Each participant will keep a 24h food record

preceding the testing trials so they have the ability to replicate the exact methodology for each subsequent

test. All of the participants will be asked to stay hydrated leading up to each trial and will be instructed to

refrain from strenuous activity, caffeine and alcohol 24h preceding testing sessions. Each individual will

maintain similar levels of activity (volume and intensity) and exercise training 8 days preceding the trials

while following the supplement regimen. Exercise tests will be performed at the same time of day, each

testing day, as closely as possible.


Protection Measures: All participants will be fully informed of all study procedures, and may withdraw

at any time. All physiological testing will be completed in the HPPE department’s Human Performance

Lab, supervised by the leading researcher, Nicolas Aguila, and one of the assisting researchers (Dr.

Robinson) who is ACSM certified (American College of Sports Medicine). Additionally, all other testing

trials will be completed on an indoor turf field, supervised by the participant’s soccer coach and the other

assisting researchers. Confidentiality and anonymity of participants will be maintained consistently

throughout the study. Consent forms and raw data will be stored in a secure location that can only be

accessed by the researchers. Participants will also be asked to fill out a PAR-Q questionnaire regarding

their health status prior to any testing, and if necessary, have a physician clearance before participating in

the study. Results of the study will be reported as group data, without any individual subject identifying

information.

Consent: Participants will be asked to read over and sign the consent form before any testing commences.

The informed consent is attached separately.

Changes : If any changes are made to the research I will contact the IRB immediately and fill out the

needed paperwork.

____________________________________________________ ___________________

Name and Signature of Department Chair or Appropriate Person Date

____________________________________________________ ___________________

Name and Signature of IRB chair Date Date

Documents

Final Thesis Paper