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The effect of beetroot juice on exercise performance in normoxia and moderate hypoxia MacLeod, Kristin Elizabeth 2013

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  THE EFFECT OF BEETROOT JUICE ON EXERCISE PERFORMANCE IN NORMOXIA AND MODERATE HYPOXIA    by   KRISTIN ELIZABETH MACLEOD      A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF    MASTER OF SCIENCE   in   THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES  (Kinesiology)    THE UNIVERSITY OF BRITISH COLUMBIA  (Vancouver)      December 2013   ? Kristin Elizabeth MacLeod   	 ? ii	 ?Abstract  The effect of beetroot juice on exercise tolerance in normoxia and moderate hypoxia  AIMS: Beetroot juice (BR) has been shown to lower the oxygen cost of exercise in normoxia, and may also be beneficial to performance in hypoxia. We investigated the effect of BR on steady state economy and 10-km time trial (TT) performance in normoxia and in hypoxia (simulated altitude of 2500 m).   METHODS: Twelve trained male cyclists (VO2max ? 60 ml?kg-1?min-1) completed four exercise trials. Two hours prior to exercise, subjects consumed 70 mL BR (~6 mmol nitrate) or 70 mL placebo (nitrate-depleted BR) in a randomized, double-blind manner. Subjects then completed a 15-min self-selected cycling warm-up followed by a 15-min steady-state exercise bout at 50% of maximum power output and a 10-km time trial (TT) in either normoxia (~21% O2) or hypoxia (~16% O2). Environmental conditions were randomized and single blind.   RESULTS: Economy at 50% power output was similar in hypoxic and normoxic conditions (p > 0.05), but subjects had a significantly higher mean power output in the normoxic TT relative to the hypoxic TT (p < 0.05). BR did not affect economy, mean power output, or time to complete the 10-km TT relative to placebo in normoxia (p > 0.05 in all comparisons). Similarly, in hypoxia, BR did not affect economy, steady state SpO2, mean power output or time to complete the 10-km TT relative to placebo (p > 0.05 in all comparisons). BR supplementation resulted in a significantly greater fraction of expired nitric oxide relative to PL (28 [12] vs. 22 [9] ppb).   CONCLUSIONS: In a small sample of well-trained male cyclists, BR did not improve exercise performance in normoxia or hypoxia. This finding is possibly related to BR dosage, although it is congruent with previous evidence showing that BR may be less effective in trained populations in normoxia. The lack of an effect in hypoxia was contrary to our hypothesis.	 ?       	 ? iii	 ?Preface Collaborators and co-authors of this thesis study are as follows:  ? Dr. James Rupert, PhD, supervised this project, assisted in the ethics application, helped with the funding application to Own the Podium (OTP), and provided guidance during sample analysis and writing. ? Dr. Michael Koehle, MD, PhD, aided in the development of the research design and assisted with equipment trouble-shooting and data analysis. ? Dr. Benjamin Sporer, PhD, assisted in development of the research design, provided insight into the practical application of research and helped with the application for funding through OTP. ? Dr. Susan Barr, PhD, assisted in the development of the research design from a sport nutrition standpoint. ? Martin MacInnis, BSc, assisted in the development of the research design, ethics approval, chamber reconfiguration, data collection, data analysis, and provided feedback during the writing process. ? Sean Nugent assisted in data collection. ? Kirsten Hogg, PhD, assisted with plasma nitrate and nitrite assays for plasma sample analysis ? Kristin MacLeod, BSc, developed the research design, obtained funding from OTP and the UBC Faculty of Education, completed the application for ethics approval, recruited subjects, performed data collection, assisted with assay analysis, analyzed data and wrote the final thesis document.  No manuscripts resulting from the work presented in this thesis have been published to date. The abstract and poster attached in Appendix A were accepted for a poster presentation at the Sport Innovation (SPIN) Summit organized by OTP in September 2013, in Calgary, Canada.  This study involved human subjects and was granted full board approval from the University of British Columbia Clinical Research Ethics Board (H12-01832). 	 ? iv	 ?Table of contents  Abstract	 ?.......................................................................................................................................	 ?ii	 ?Preface	 ?........................................................................................................................................	 ?iii	 ?Table	 ?of	 ?contents	 ?....................................................................................................................	 ?iv	 ?List	 ?of	 ?tables	 ?.............................................................................................................................	 ?vi	 ?List	 ?of	 ?figures	 ?...........................................................................................................................	 ?vii	 ?List	 ?of	 ?abbreviations	 ?...........................................................................................................	 ?viii	 ?Acknowledgements	 ?................................................................................................................	 ?ix	 ?1.	 ?Introduction	 ?..........................................................................................................................	 ?1	 ?1.1 Overview	 ?..........................................................................................................................................	 ?1	 ?1.2 Aerobic exercise	 ?..............................................................................................................................	 ?1	 ?1.2.1	 ?Adaptations	 ?to	 ?endurance	 ?exercise	 ?...................................................................................................	 ?2	 ?1.2.3	 ?Determinants	 ?of	 ?performance	 ?.............................................................................................................	 ?3	 ?1.2.4	 ?Limitations	 ?to	 ?aerobic	 ?capacity	 ?and	 ?performance	 ?.....................................................................	 ?5	 ?1.2.5	 ?Aerobic	 ?exercise	 ?in	 ?hypoxia	 ?...................................................................................................................	 ?5	 ?1.2.6	 ?Effects	 ?of	 ?altitude	 ?training	 ?....................................................................................................................	 ?6	 ?1.3 Exercise and nitrate	 ?........................................................................................................................	 ?7	 ?1.3.1	 ?Nitric	 ?oxide	 ?pathways	 ?.............................................................................................................................	 ?9	 ?1.3.2	 ?Sex	 ?differences	 ?in	 ?response	 ?to	 ?nitrate	 ?supplementation	 ?........................................................	 ?11	 ?1.3.3	 ?Effect	 ?of	 ?beetroot	 ?juice	 ?on	 ?exercise	 ?..................................................................................................	 ?11	 ?1.3.4	 ?Nitrate	 ?supplementation	 ?and	 ?normoxic	 ?exercise	 ?performance	 ?..........................................	 ?13	 ?1.3.5	 ?Beetroot	 ?juice	 ?and	 ?hypoxic	 ?performance	 ?studies	 ?......................................................................	 ?14	 ?1.3.6	 ?Nitric	 ?oxide	 ?in	 ?hypoxia	 ?..........................................................................................................................	 ?14	 ?1.4 Summary and potential significance	 ?..........................................................................................	 ?16	 ?2.	 ?Effect	 ?of	 ?beetroot	 ?juice	 ?on	 ?exercise	 ?performance	 ?in	 ?normoxia	 ?and	 ?moderate	 ?hypoxia	 ?.....................................................................................................................................	 ?17	 ?2.1 Introduction	 ?..................................................................................................................................	 ?17	 ?2.2 Purpose, objectives and hypotheses	 ?...........................................................................................	 ?18	 ?2.3 Methods	 ?........................................................................................................................................	 ?19	 ?2.3.1	 ?Participant	 ?information	 ?.......................................................................................................................	 ?19	 ?2.3.2	 ?Experimental	 ?design	 ?..............................................................................................................................	 ?19	 ?2.3.3	 ?Statistical	 ?analysis	 ?..................................................................................................................................	 ?25	 ?2.4 Results	 ?..........................................................................................................................................	 ?26	 ?2.4.1	 ?Subject	 ?characteristics	 ?..........................................................................................................................	 ?26	 ?2.4.2	 ?Hypoxic	 ?and	 ?normoxic	 ?exposures	 ?.....................................................................................................	 ?27	 ?2.4.3	 ?Resting	 ?blood	 ?pressure	 ?and	 ?FENO	 ?....................................................................................................	 ?27	 ?2.4.4	 ?Steady-??state	 ?cycling	 ?...............................................................................................................................	 ?29	 ?2.4.5	 ?10-??km	 ?time	 ?trial	 ?.......................................................................................................................................	 ?29	 ?2.5 Discussion	 ?....................................................................................................................................	 ?33	 ?2.5.1	 ?Layout	 ?of	 ?discussion	 ?...............................................................................................................................	 ?33	 ?2.5.2	 ?Pre-??	 ?and	 ?post-??supplementation	 ?variables	 ?....................................................................................	 ?33	 ?2.5.3	 ?Blood	 ?pressure	 ?..........................................................................................................................................	 ?34	 ?	 ? v	 ?2.5.4	 ?Exercise	 ?economy	 ?....................................................................................................................................	 ?35	 ?2.5.5	 ?10-??km	 ?TT	 ?performance	 ?.........................................................................................................................	 ?38	 ?2.5.6	 ?Summary	 ?.....................................................................................................................................................	 ?41	 ?3.	 ?Conclusion	 ?..........................................................................................................................	 ?42	 ?3.1 Conclusions regarding thesis hypotheses	 ?.................................................................................	 ?42	 ?3.2 Strengths, limitations, future directions	 ?....................................................................................	 ?43	 ?Bibliography	 ?...........................................................................................................................	 ?45	 ?Appendix	 ?A	 ?..............................................................................................................................	 ?54	 ?Appendix	 ?B	 ?..............................................................................................................................	 ?56	 ?Appendix	 ?C	 ?..............................................................................................................................	 ?62	 ?Appendix	 ?D	 ?..............................................................................................................................	 ?64	 ?Appendix	 ?E	 ?..............................................................................................................................	 ?68	 ?Appendix	 ?F	 ?..............................................................................................................................	 ?71	 ?	 ?            	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ? vi	 ?List of tables 	 ?Table 1: Inclusion and exclusion criteria.  ........................................................................ 19	 ?	 ?Table 2: Summary of procedures for all subjects on laboratory visits.  ........................... 25	 ?	 ?Table 3: Subject anthropometric, fitness, and power data.  .............................................. 27	 ?	 ?Table 4: Summary data for blood pressure and fraction of expired nitric oxide pre- and post- supplementation.  ..................................................................................................... 28	 ?	 ?Table 5: Summary statistics for systolic and diastolic blood pressures and fraction of expired nitric oxide ........................................................................................................... 31 	 ?Table 6: Summary statistics of physiological variables during 50% steady state cycling. ........................................................................................................................................... 32	 ?	 ?Table 7: Summary statistics of physiological variables during the 10-km time trial. ...... 32	 ?	 ?Table 8: Summary of studies investigating the effects of beetroot supplementation. ...... 56	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ? vii	 ?List of figures  Fig 1: Nitric oxide formation. ........................................................................................ 10   Fig 2: General study overview. ...................................................................................... 21  Fig 3: Breathing set-up. .................................................................................................. 23  Fig 4: Timeline of data collection sessions (2-5). .......................................................... 26  Fig 5: Systolic and diastolic blood pressure pre- and post-supplementation. ................ 28  Fig 6: Economy during 50% steady state cycling. ......................................................... 29  Fig 7: Mean power output during the 10-km time trial. ................................................ 30  Fig 8: Heart rate during 50% steady state cycling. ........................................................ 68  Fig 9: SpO2 during 50% steady state cycling. ................................................................ 68  Fig 10: Pulmonary ventilation during 50% steady state cycling. .................................. 69  Fig 11: Individual subject economy data during 50% steady state cycling in normoxia.  ........................................................................................................................................ 69  Fig 12: Individual subject economy data during 50% steady state cycling in hypoxia.  ........................................................................................................................................ 70  Fig 13: Time to complete the 10-km time trial. ............................................................. 71  Fig 14: Individual time to complete the 10-km time trial in normoxia. ........................ 71  Fig 15: Individual time to complete the 10-km time trial in hypoxia. ........................... 72  Fig 16: Individual subject data for mean power output during the 10-km time trial in normoxia. ....................................................................................................................... 72  Fig 17: Individual subject data for mean power output during the 10-km time trial in hypoxia. .......................................................................................................................... 73     	 ? viii	 ?List of abbreviations    ADP: adenosine diphosphate  ATP: adenosine triphosphate  BP: blood pressure  -DBP: diastolic blood pressure  -SBP: systolic blood pressure  BR: beetroot juice  cGMP: cyclic guanosine monophosphate  CREB: Clinical Review Ethics Board  FENO: fraction of expired nitric oxide  HR: heart rate  L-NAME: L-NG nitroarginine methyl ester  NIRS: near-infrared spectroscopy  NO: nitric oxide  NO2-: nitrite  NO3-: nitrate NOS: nitric oxide synthase (pathway)  PCr: phosphocreatine   Pi: inorganic phosphate  PL: placebo  PO2: partial pressure of oxygen  SD: standard deviation  SpO2: pulse oxygen saturation  TOI: tissue oxygenation index  TT: time trial  UBC: University of British Columbia   VE: pulmonary ventilation  O2: oxygen uptake  O2max: maximal oxygen uptake  W: watts              ?V?V	 ? ix	 ? Acknowledgements  This project was realized thanks to financial support from Own the Podium (OTP) and the University of British Columbia Faculty of Education.  I would not have been able to complete this project without support from my supervisor, committee, lab mates, friends and family. I would like to thank:  ? My supervisor, Dr. James Rupert ? My committee members, Dr. Michael Koehle, Dr. Benjamin Sporer and Dr. Susan Barr ? All of the athletes who participated in this study ? Own the Podium and the University of British Columbia Faculty of Education ? Dr. Trent Stellingwerff  ? Cycling Canada, Rowing Canada, Athletics Canada and Canada Snowboard ? Martin MacInnis and Sean Nugent ? Dr. Kirsten Hogg ? Dr. Keith Lohse ? Dr. Bill Sheel and Paolo Dominelli ? Sarah Koch, Maha Elashi, Beverly Larssen, Eric Carter          	 ? 1	 ?1. Introduction  1.1 Overview	 ?At altitude, decreases in the barometric pressure and the alveolar partial pressure of oxygen result in less oxygen reaching the tissues (reviewed in Sheel et al., 2010). The drop in available oxygen detrimentally affects aerobic capacity. For example, O2max decreases by ~6%/1000 m between 300 m and 2800 m above sea level (Wehrlin and Hall?n 2006). These factors combine to reduce overall training quality, particularly with acute hypoxic exposure in absence of acclimatization. As athletes often train and compete at moderate altitude (i.e., 1500-2500 m), the ability to maintain high exercise intensity is important for performance.  Beetroot juice (BR), a supplement with a naturally high concentration of nitrate (NO3-) has gained popularity with endurance athletes aiming to improve performance. There is evidence to suggest that dietary NO3- improves oxygen delivery (Masschelein 2012), lowers blood pressure during exercise recovery (Larsen et al., 2010; Kenjale et al., 2011), and increases mitochondrial efficiency through an improved oxidative phosphorylation ratio (Larsen et al., 2011). When oxygen is less available from the environment (e.g. at altitude), BR may have even greater physiological effects (Dauncey 2012); however, the efficacy of acute BR supplementation for exercise performance in moderate hypoxia requires further research. As hypoxia limits aerobic capacity, the potential benefits of BR are particularly relevant to aerobic exercise.     1.2 Aerobic exercise	 ?Aerobic exercise, which involves continuous whole body or large muscle mass exercise, stresses the cardiorespiratory system. Generally, moderate intensity constant-load exercise can be sustained for relatively long periods of time due to the predominant use of fatigue resistant slow-twitch muscle fibres. These fibres have a slower contraction speed, meaning they utilize ATP at a slower rate; therefore, sufficient ATP production can be maintained through aerobic metabolism (McArdle et al., 2007). Over time, the ?V	 ? 2	 ?result of endurance training is increased aerobic capacity, or an improved ability to maintain a given power output or velocity (Jones and Carter 2000). Improved aerobic capacity is indicative of better endurance performance (reviewed in Jones and Carter 2000), making improved aerobic capacity a central goal of many training programs. Exercise causes an acute stress response throughout the body. At rest, energy production requires oxygen, which is brought in through the lungs and diffuses to the blood within the pulmonary vasculature. The oxygenated blood flows back to the heart where it is pumped into the systemic circulation (McArdle et al., 2007) where the oxygen diffuses into the tissue. Exercise increases the demand for oxygen, and the body responds with an increase in tidal volume, breathing frequency, heart rate and stroke volume, all of which help to maintain sufficient blood flow and oxygen delivery to support the working muscles (West 2007). Improved oxygen delivery is supported by increased extraction at the tissue level, which also facilitates the maintenance of elevated energy production. During aerobic exercise the main energy pathway is oxidative phosphorylation. At low, continuous exercise intensities, fat is the primary fuel for energy production, while higher intensities rely predominantly on carbohydrate metabolism (McArdle et al., 2007). Over time, these acute responses to exercise result in chronic training adaptations that improve the body?s ability to maintain a work rate substantially above resting rates over prolonged periods.  1.2.1 Adaptations to endurance exercise  Aerobic training causes both peripheral and central adaptations that act to enhance oxygen delivery to the mitochondria as well as enhance the ability of muscles to use the available oxygen (McArdle et al., 2007). The training-induced increase in plasma volume and red blood cell mass increases the volume of blood passing through the heart, which results in cardiac remodeling?most notably physiological hypertrophy of the left ventricle?to help reduce strain on the myocardium during exercise (Ellison et al. 2011). In trained athletes, increased stroke volume accounts for most of the increase in cardiac output, as maximal heart rate is often unchanged or lower in comparison to untrained individuals (Wilmore et al., 2008). The increased cardiac output allows for increased perfusion of exercising muscles. At the muscle level, there is increased capillarization to accommodate greater blood flow and increased myoglobin content to improve oxygen 	 ? 3	 ?delivery to the mitochondria. Greater energy demand on muscles requires a commensurate increase in energy production. The increase in energy production is facilitated by greater mitochondrial density and size (Wilmore et al., 2008). Chronic endurance exercise has also been found to cause vascular remodeling, resulting in widened vessels and increased vascular tone (Niebauer and Cooke 1996). High shear stress promotes secretion of vasodilators (Paszkowiak and Dardik 2003) that are important for maintaining blood pressure. Highly trained athletes repeatedly experience high shear stress due to the increased cardiac output and hyperemic response associated with exercise. Increased shear may result in improved bioactivity of nitric oxide (Green et al., 2004). Changes in pulmonary function are important contributors to adaptations elsewhere in the body. Trained individuals have a lower ventilation at a given submaximal exercise intensity, coupled with a much greater maximal ventilation than untrained individuals (McArdle et al., 2007). Pulmonary blood flow increases during high intensity exercise. Together, the increase in blood flow and ventilation improve pulmonary diffusion of oxygen (Wilmore et al., 2008). Once oxygen is carried to the muscle, there is greater oxygen extraction at the tissue level in trained individuals, a result of increased tissue perfusion (Bassett and Howley 2000).  Aerobic exercise causes adaptation across all body systems. These changes improve the body?s ability to maintain continuous exercise at a given intensity and increase the workload the body can produce for extended durations by increasing oxygen delivery and extraction. Overall, these adaptations contribute greatly to cardiorespiratory health and improve endurance performance.   1.2.3 Determinants of performance For high-level athletes, the main goals of endurance training are to improve aerobic capacity and performance. Performance may be measured in many ways, including competition results, time to complete a given task, and mean power output during an event. Endurance performance potential is often predicted from physiological tests that provide insight into an athlete?s aerobic capacity (e.g., exercise economy, and 	 ? 4	 ?maximal oxygen uptake [ O2max]). O2max and exercise economy are described in more detail below.  Maximal oxygen uptake (??? ? ?? )  O2max is the highest rate at which an individual can use oxygen during maximal exercise (Jones and Carter 2000). Both athletes and exercise physiologists consider O2max the ?gold-standard? measurement of aerobic capacity. Generally, individuals who train in endurance events will have a higher O2max than their anaerobic sport or untrained counterparts. Elite athletes will also generally have a higher O2max than sub-elite athletes. While a good measurement of aerobic fitness and a general marker of performance potential (Levine 2008), O2max is not necessarily a predictor of performance, particularly among training-matched athletes. In other words, a high O2max is necessary to perform well, but in a group of individuals with similar O2max values, there can be a large variation in performance, suggesting that there are many additional factors (both physiological and psychological) that affect performance.   Exercise economy  Exercise economy (often grouped with mechanical efficiency) is a measure of the oxygen cost for a given amount of physiological work. During exercise on a cycle ergometer, O2 increases by ~10 mL?min-1 for each additional watt of external power output (Jones et al., 2013). While economy improves with training (i.e., lowered oxygen cost of exercise), often as a result of training adaptations and improved biomechanical efficiency, it does not change greatly over time in trained individuals.  Training may increase mitochondrial density and therefore energy production. For example, the vastus lateralis muscle of highly trained cyclists typically has a higher percentage of Type I muscle fibres (Coyle et al., 1992), which have a greater mitochondrial density than Type II fibres and are more efficient for continuous energy production (Joyner and Coyle 2008). Irrespective of mitochondrial density, energy required for a given workload should be fairly consistent within an individual, and independent of how efficiently the energy is produced. In trained athletes, economy will ?V ?V?V ?V?V ?V?V ?V?V?V	 ? 5	 ?not differ greatly over time (assuming training is maintained), although economy may improve as O2max declines with age (Jones 1998). Exercise economy may be a better predictor of performance in athletes closely matched in training load and O2max: O2max is a ceiling value for the rate of oxygen use, but economy determines how much work can be performed with that oxygen. 1.2.4 Limitations to aerobic capacity and performance  Aerobic capacity is limited by anything that negatively affects oxygen delivery to, or oxygen extraction by, exercising muscles. These factors may include anything that detrimentally affects pulmonary diffusion capacity (e.g., ventilation/perfusion mismatch or shunts), cardiac output (e.g., decreased stroke volume), oxygen carrying capacity (e.g., low hemoglobin mass), or exercising muscles (e.g., decreased mitochondrial enzyme activity) (Bassett and Howley 2000). A gas exchange limitation will likely impact highly trained athletes to a greater extent than recreationally active individuals (potentially due to a greater total blood volume and cardiac output), resulting in the muscular demand for oxygen outstripping delivery at high exercise intensities. For instance, hypoxic exposure negatively affects gas exchange and aerobic performance. A drop in the partial pressure of oxygen decreases availability of oxygen to the body. By extension, hypoxia causes a resultant decline in aerobic capacity that limits endurance performance. This drop is particularly relevant to athletes who attend training camps or competitions at moderate altitude, where training intensity and recovery will likely differ from sea level.  1.2.5 Aerobic exercise in hypoxia A drop in PO2 decreases the body?s ability to deliver saturated hemoglobin to the tissues and the capacity for oxygen to diffuse to the mitochondria (Storz et al., 2011). The body exhibits a number of physiological adjustments in response to hypoxia in an attempt to maintain oxygen supply. The immediate response to hypoxia is increased ventilation to maintain oxygen saturation, resulting in an increase in exhaled CO2, and subsequent respiratory alkalosis (B?rtsch and Saltin 2008). This ventilatory response shifts the oxygen dissociation curve to the left, which better allows for oxygen to bind to hemoglobin due to an increased blood-O2 affinity (West 2007). Cardiac output also increases (via elevated heart rate) to balance oxygen delivery with demand (Casey and ?V ?V ?V	 ? 6	 ?Joyner 2012); however, maximal heart rate may be reduced in hypoxia, as is maximal cardiac output (B?rtsch and Saltin 2008). Furthermore, an attenuated sympathetic response in contracting muscle, despite an increase in vasoconstrictor signals, results in compensatory vasodilation (Casey and Joyner 2012). Despite compensations, if oxygen saturation is decreased, blood flow during maximal large-muscle mass exercise is lower than in normoxic exercise (Calbet 2002). Reduced blood flow limits oxygen delivery, and O2max decreases by ~6%/1000 m altitude (to 3000 m, after which the rate of decrease may increase), due to the drop in oxygen driving pressure (Wherlin and Hall?n 2006). Even with the reduced blood flow, there is evidence that pulmonary and muscle diffusion are the main components affecting this decrease in aerobic capacity (Wagner 2010). In the practical sense, physiological adaptations that allow an individual to maintain a high aerobic capacity will benefit endurance exercise at altitude.  Training intensity and endurance sport performance are both affected by hypoxia, which has prompted athletes to try different strategies to attenuate the decline in physiological function. It has been shown repeatedly that individuals with higher baseline fitness tend to have a greater decline in aerobic performance than the untrained (Fulco et al., 1998). Even for athletes who are well matched for fitness at sea level, there is great inter-individual variation for response to hypoxia.  Although O2max drops significantly, economy remains consistent after hypoxic acclimatization at moderate altitudes, although there may be modest improvements (Lundby et al., 2006). This is potentially because a given workload requires an absolute O2 that remains constant. However, it is more difficult (i.e. requires more work) to maintain a given workload at altitude?for example, pulmonary ventilation increases?so the body must work comparatively harder in acute hypoxia than at sea level.   1.2.6 Effects of altitude training  Although altitude reduces aerobic capacity, acclimatization improves performance at altitude and may have beneficial effects on sea-level performance. However, there is great individual variation in the extent of these responses to both acute and longer-term exposures (Martin et al., 2010). A number of different strategies are used to elicit this ?V ?V?V	 ? 7	 ?benefit. For instance, living ?high? and training ?low? is a popular approach, combining the benefit of training at sea-level with the physiological responses to prolonged altitude exposure during rest that could benefit endurance exercise (Millet et al., 2010). The efficacy of such strategies is highly variable and seems to depend on each individual?s ability to acclimatize.  Ideally, endurance athletes competing at altitude will be able to acclimatize fully to the conditions in which they will compete (a recommended two weeks for venues up to 3000 m) (Bergeron et al., 2012); however, even with acclimatization, performance at altitude will not equal sea-level capabilities in endurance sport (if aerodynamic drag is not factored in). Moreover, acclimatization is not always feasible, nor optimal for training, as athletes are unable to maintain training intensities comparable to sea level at altitude (Millet et al., 2010) and may have busy competition schedules, restricting the time they could spend at altitude before a single event.  To augment training efforts, athletes often supplement with dietary or pharmaceutical aids. Various treatments have been investigated to alleviate the strain of exercise in hypoxia. For example, sildenafil results in reduced hypoxia-induced pulmonary hypertension (Zhao et al., 2001; Ghofrani et al., 2004), while improving exercise capacity at altitude through improved oxygen saturation and increased maximal workload and cardiac output (Ghofrani et al., 2004). There are concerns regarding the use of sildenafil at altitude, as it may increase the severity of headaches, a well-documented symptom of acute mountain sickness (Hackett and Roach 2001). Athletes could also experience other undesirable side effects (e.g., blue tinted vision (Marmor and Kessler 1999)).   1.3 Exercise and nitrate	 ?In sport culture, supplementation is a common tactic to improve performance. In recent years, research into nitrate (NO3-) and nitrite (NO2-) supplementation has become more popular, particularly involving the use of beetroot juice (BR) as a source of NO3- leading to nitric oxide (NO) production. In an applied setting, it has become common for athletes to use BR for training and competition. These supplements contain juice from beets (often exclusively), and vary in volume and NO3- content, depending on whether 	 ? 8	 ?the supplement is ?concentrated? or not. For example, a BR ?sport shot? used in studies contains 70 mL of concentrated BR and ~4.2 mmol of NO3- (Wylie et al., 2013). Some studies, however, supplement with up to 500 mL of BR a day, with the maximum reported dose being 500 mL BR (containing ~11 mmol NO3-) administered daily over a period of six days (Bailey et al., 2009). While studies of BR and exercise capacity in both clinical populations and averagely active individuals are promising (e.g., Kenjale et al., 2011), the few studies conducted in highly trained athletes in normoxia have returned mixed results (reviewed in Hoon et al., 2013b). The few studies of BR in hypoxia have been unanimous in demonstrating positive effects (Vanhatalo et al., 2011; Masschelein et al., 2012; Muggeridge et al., 2014), although only one of these studies (Muggeridge et al., 2014) has been completed with well trained athletes. Current evidence suggests that training and health status, sex (Kapil et al., 2010), dosage (Wylie et al., 2013a) and altitude (Dauncey 2012) may all play a large role in the efficacy of BR as a supplement. Therefore, it is important to test the usefulness of BR in highly trained endurance athletes and to determine the conditions in which it is most effective.  In trained endurance athletes, there is evidence for responders and non-responders to BR supplementation (e.g., Christensen et al., 2012). Varied ?response? between individuals may be related to differences in adaptations to the vasculature, endothelial function, and aerobic system. Physiological adaptations of endurance exercise should result in excellent oxygen delivery, potentially masking or making redundant any BR benefit. This possibility is reflected in a study finding no benefit of a six-day BR supplementation for a 400-kcal (~18 minute) time trial (TT) in elite male cyclists ( O2max 72.1 [4.5]) in normoxia (Christensen et al., 2012). This finding is contrasted by data from Lansley et al. (2011a), which demonstrated a significant improvement in 4- and 16.1-km TT performance in club-level males ( O2max 56 [5.7] ml?kg-1?min-1) following acute BR supplementation. A meta-analysis by Hoon et al. (2013b) indicated greater performance improvements from BR have been found in inactive or recreationally active individuals. As yet, there are no studies to indicate that so-called ?responders? would continue to respond repeatedly.  Despite differences in the effect of BR among athletes, some of the factors contributing to aerobic performance are limited in acute hypoxia, making it plausible that ?V?V	 ? 9	 ?BR may enhance exercise tolerance even in those individuals who do not respond to BR supplementation at sea level; however, this may be confounded by the variable response to hypoxia alone. The background physiology and research are presented below in more detail. Studies conducted to date assessing the efficacy of BR supplementation are summarized in Table 8 in Appendix B.  1.3.1 Nitric oxide pathways Nitric oxide (NO) is a signaling molecule and potent vasodilator formed by nitric oxide synthase (NOS) enzymes (constitutive, inducible or neuronal) in response to chemical and mechanical stimuli (MacMicking et al. 1997; Bogdan 2001) through the oxidation of L-arginine (Palmer et al., 1988). This complex reaction is catalyzed by the NOS enzymes and requires oxygen and other substrates (Lundberg and Weitzberg 2010; Bailey et al., 2012).  A secondary pathway for the formation of NO has been suggested, in which dietary nitrate (NO3-) is reduced to nitrite (NO2-) by anaerobic bacteria in the mouth (reviewed in Lundberg et al., 2008). Some of this NO2- is further reduced to NO in the acidic environment of the stomach (Lundberg and Govoni 2004; Lundberg et al., 2008), while other NO2- molecules continue to circulate in the blood and may form NO systemically, catalyzed by hemoglobin, myoglobin, and xanthine oxidoreductase among other species (Lundberg et al., 2011b; Lundberg et al., 2008). When the resultant NO from either pathway diffuses into surrounding smooth muscle cells, it activates guanylyl cyclase, which in turn stimulates cyclic guanosine monophosphate (cGMP) formation and smooth muscle cell relaxation (Bredt and Snyder 1994). See Figure 1.    	 ? 10	 ? Fig 1: Sources of nitric oxide (NO) in the human body. NO can form in the body through the nitric oxide synthase pathway (NOS) or through nitrate (NO3-) in the diet. NO3- may be reduced to nitrite (NO2-) and subsequently NO. NO may also be oxidized to form NO2- and NO3-.  NO is known to be particularly important for health. In addition to being a vasomodulator, NO plays roles in cellular respiration and inflammation (Lundberg et al., 2008). For example, in male Munich Wistar rats, chronic (two month) blocking of the NOS pathway through the use of L-NG nitroarginine methyl ester (L-NAME) resulted in systemic hypertension and glomerular injury (Baylis et al., 1992). Jones et al. (2004) further reported that in humans, acute L-NAME infusion increased mean arterial pressure, and decreased O2max and heart rate.  Inorganic NO3- and BR supplementation have been found to decrease blood pressure (BP) (Siervo et al., 2013). Specifically, BR supplementation has been shown to lower diastolic blood pressure (DBP), systolic blood pressure (SBP), and mean arterial pressure (e.g., Webb et al., 2008). These results coincide with greater plasma [NO2-] following supplementation. An increase in plasma [NO2-] may be the result of NO3-reduction, indicating that NO3- supplementation is responsible for subsequent BP reductions, possibly mediated by increased NO. Chronic intake of a nitrate-rich diet (e.g., the ?Mediterranean Diet?) may have a cardioprotective effect, suggesting long-term ?V	 ? 11	 ?benefit  (Hu 2003; Lundberg et al., 2006; Lidder and Webb 2012). Decreased BP is maintained for at least 15 days with chronic BR supplementation (Vanhatalo et al., 2010), but there are no investigations of longer duration. There may be a dose-dependent relationship between NO3- intake and change in BP (2010) (Kapil et al., 2010). An increase in BP occurred when oral microflora were disrupted with the use of antibacterial mouthwash (Kapil et al., 2012), further demonstrating the significance of the oral pathway in generating NO.  1.3.2 Sex differences in response to nitrate supplementation Sex differences in the effect of NO3- supplementation on BP may exist. Kapil et al., (2012) reported that premenopausal females had higher basal [NO2-] coupled with lower BP than age-matched males and, following supplementation with potassium nitrate males had a greater reduction in BP than females. Thoonen et al. (2012), suggested that nitrate supplementation may be less effective in women, due to possible differences in sex-specific NO-cGMP signaling. The reason for the discrepancy is unknown but may be caused by differences in vasodilation signals. For example, it has been suggested that in female mice the main endothelial-derived relaxing factor is endothelial-derived hyperpolarizing factor, while in males it is NO and prostacyclin (Scotland et al., 2005). The possibility that supplement efficacy varies between sexes warrants further research, as data demonstrating such a dichotomy would be of interest to athletes interested in nitrate supplementation,   1.3.3 Effect of beetroot juice on exercise  Bailey et al. (2010) demonstrated that BR supplementation before small-muscle exercise significantly altered the intramuscular environment (demonstrated by decreased ADP and Pi accumulation and decreased PCr depletion) and increased exercise tolerance in normoxia, relative to placebo. In this study, subjects completed incremental double-legged knee extension exercise in a step-wise manner until task failure. There was a 25% increase in time to task failure with BR, with each of the seven subjects performing better in the BR condition. The authors suggest that this finding may be the result of a 	 ? 12	 ?decreased total ATP cost for muscle force production, as reduced ADP and Pi accumulation may be due to coupling between ATP hydrolysis and the energy required for force production at a given work rate with BR supplementation. If ADP and Pi are reduced, oxidative phosphorylation would likely also be reduced (Brown 1992). Results from this study indicated improved matching of energy produced for energy required (Bailey et al., 2010). The majority of BR studies in humans have investigated performance outcomes rather than the effect?s mechanism. Studies in animal models have allowed for a more in depth understanding of how BR and NO3- supplementation may affect muscle function. For example, fast-twitch muscle fibres from C57bl/6 male mice supplemented with 1 mM sodium nitrate in water for seven days had increased Ca2+ handling proteins and myoplasmic free [Ca2+], as well as increased contractile force and a faster rate of force development compared to untreated mice (Hern?ndez et al., 2012). Relatedly, Ferguson et al. (2012), demonstrated improved skeletal muscle blood flow and vascular conductance in male Sprague-Dawley rats supplemented with 1 mmol NO3-?kg-1?day-1 in BR during treadmill running, primarily in Type IIB+d/x fibres. Together, these studies indicate a greater role for NO in oxygen matching in fast-twitch fibres. Previous findings that NO directly inhibits force-generating proteins in skeletal muscle myofibrils (Galler et al., 1997) may seem counterintuitive considering conclusions by both Hern?ndez et al., (2012) and Ferguson et al., (2012). However, this study used muscle fibres from rat leg muscles such as the soleus and vastus lateralis, which, in endurance trained humans, are likely composed of a high proportion of Type 1 fibres (Coyle et al., 1992). These results as a whole seem to be supported in humans by Bailey et al. (2010), as discussed above. It has also been demonstrated in male Sprague-Dawley rats that renal injury was attenuated after a medium-to-high dose of dietary NO2- was provided in drinking water concomitantly to chronic L-NAME infusion (Kanematsu et al., 2008). This provides evidence that the NOS and NO3- ? NO2- ? NO systems are independent, and one can act to maintain NO production should the other be compromised. This has important clinical applications, but also strengthens the rationale for BR as a supplement in hypoxia.  	 ? 13	 ?1.3.4 Nitrate supplementation and normoxic exercise performance  Although most human research has been conducted using untrained populations, a small, but highly relevant, number of studies have investigated the effect of BR on endurance performance in highly trained athletes. Studies of highly trained or elite cyclists in normoxia have found improvement in time trial performance following BR supplementation in a 4-km TT (Lansley et al., 2011a), a 10-km TT (Cermak et al., 2012a), and a 16.1-km TT (Lansley et al., 2011a) but not in a 50-mile TT (Wilkerson et al., 2012). Lansley et al. (2011a) concluded that mean power output increased for both 4-km and 16.1-km TT distances with BR supplementation, although VO2 values did not differ between BR and placebo (PL). Conversely, Wilkerson et al. (2012) demonstrated that neither power output nor mean O2 differed significantly between BR and PL trials. This study reported a lower mean O2 with BR compared to placebo over the 50-mile TT that approached statistical significance (p = 0.06), which may be practically meaningful. Wilkerson et al. (2012) also demonstrated that individuals with higher plasma [NO2-] following supplementation performed better in the time trial. While the 50-mile TT completed in this study was of a much longer distance than those used in other studies (e.g., Lansley et al. (2011a); Cermak et al. (2012a)), the exercise intensity was fairly low. This may have limited the efficacy of the NO3- ? NO2- ? NO pathway, which is potentiated in hypoxic and acidic conditions (Dauncey 2012; Modin et al., 2001).  BR supplementation may be more effective for improving performance over moderate distances and/or higher intensity exercise. In a study by Christensen et al. (2012), a six-day BR loading protocol did not improve endurance capacity or repeated sprint performance in elite male cyclists. However, two of the ten subjects were identified as ?responders? who demonstrated decreased O2 at stages of 50% and 70% of aerobic peak power with BR supplementation. Furthermore, studies have concluded that an improvement in economy (reflected in decreased VO2 for a given power output) is likely due to changes in skeletal muscle rather than cardiopulmonary variables (Bailey et al., 2009; Bailey et al., 2010).   ?V?V?V	 ? 14	 ?1.3.5 Beetroot juice and hypoxic performance studies  As of 2013, data has been published from three studies that examined BR supplementation in normobaric hypoxia. Masschelein et al. (2012) used a cycling protocol consisting of 20-minute steady state exercise at 45% of maximum power output, followed by an incremental test to exhaustion. It was demonstrated that, in hypoxia (11% O2; equivalent to ~5000 m), BR increased time to exhaustion by ~5%, and improved muscle oxygenation relative to placebo. Two-legged knee extension exercise to exhaustion in hypoxia (14.5% O2) when supplemented with BR, was also demonstrated to be comparable to normoxic trials (Vanhatalo et al., 2011). Another recent study examined economy and TT performance at a simulated altitude of 2500 m (~15% O2) (Muggeridge et al., 2014) and showed that steady-state economy at 60% of maximum work rate and 16.1 km TT performance were both improved with BR, relative to placebo. This finding strengthens the rationale for BR being effective in hypoxia for highly trained individuals. In a related study, patients with peripheral arterial disease (a model of pathological ischemia that models poor oxygen delivery) demonstrated that BR supplementation increased time to onset of claudication pain (caused by too little blood flow during exercise), indicating improved perfusion in hypoxic tissue (Kenjale et al., 2011). Together, these studies suggest that exercise performance in moderate hypoxia may benefit from BR supplementation.  1.3.6 Nitric oxide in hypoxia During submaximal exercise in hypoxia, changes in oxygen saturation are compensated for by changes in cardiac output and muscle blood flow to maintain oxygen delivery (Calbet et al., 2000). However, as intensity increases during large-muscle exercise, such as cycling, the body cannot appropriately compensate for the lack of inspired oxygen. There is also a right shift of the oxygen dissociation curve that limits oxygen unloading in the muscle and decreases total oxygen consumption (Calbet et al., 2009). If cardiac output is reduced, as in maximal exercise in moderate hypoxia (Calbet et al., 2009), muscle blood flow is decreased, reducing oxygen delivery. Vasodilation may work to better match O2 requirements to delivery, regulated by oxygen sensing (Calbet et al., 2002). In hypoxia, the production of NO is up-regulated by the NOS 	 ? 15	 ?system, contributing to compensatory vasodilation and improved oxygen delivery. This up-regulation is likely related to increased shear stress and blunting of sympathetic vasoconstrictor signals (Casey and Joyner 2012). NO is also formed preferentially from NO2- as oxygen pressure falls (Cosby et al., 2003; Lundberg and Weitzberg 2005; Lundberg et al., 2008) mainly through anaerobic pathways. The reduction of NO2- is potentiated as oxygen tension falls, and the rate of conversion of NO2- to NO by hemoglobin is highest when oxygen saturation approaches 50%, due to available binding sites on hemoglobin and a peak in nitrite reductase activity (this occurs with desaturation at the tissue level) (Lundberg et al., 2008). Furthermore, this reaction benefits from a drop in pH to that expected during increased metabolic activity or hypoxia (Modin et al., 2001). Therefore, it seems possible that this secondary pathway of NO production could also improve compensatory vasodilation in hypoxia (Joyner and Casey 2013).  One of the main physiological roles played by NO is the regulation of mitochondrial respiration, making NO particularly relevant to aerobic exercise performance. For example, NO, carried by myoglobin, competes with O2 for binding sites on cytochrome c oxidase, effectively inhibiting cytochrome C oxidase and sparing O2. This results in less ATP generation in mitochondria close to the feeder artery (Dauncey 2012); however, due to a change in the inner mitochondrial membrane potential, there is less proton leak back into the mitochondrial matrix, leading to greater efficiency in cellular respiration. The O2 gradient seems to be extended, and O2 molecules spared from proximal mitochondria are used to generate ATP more distally (Dauncey 2012). Improved mitochondrial efficiency could make exercise more economical and would be particularly important in hypoxia, where matching oxygen delivery to consumption becomes more difficult. Supplementing with dietary NO3- could benefit exercise in hypoxia in a few ways: 1) improved mitochondrial efficiency (Larsen et al., 2011), meaning less O2 used for a given energy output; 2) improved muscle contractile efficiency (Bailey et al., 2010), and therefore fewer ATP used for a given force production (Jones et al., 2013); and 3) increased vasodilation due to increased NO availability, allowing for improved oxygen delivery (Siervo et al., 2013).   	 ? 16	 ? 1.4 Summary and potential significance	 ? Athletes may travel to altitude for training camps or competitions. There are currently many strategies to enhance training and performance under hypoxia, although research shows inconsistent results, due to differences in length of hypoxic exposures and dose. It is also likely there is considerable variation between individuals in the physiological response to hypoxia. Dietary NO3- in the form of BR has been demonstrated to improve normoxic exercise tolerance in clinical populations and recreational athletes. There is further evidence that exercise tolerance in simulated hypoxia may benefit from BR. Comparing recreationally active individuals and elite athletes, greater performance improvements have been demonstrated in less fit populations. In the few studies that have assessed the efficacy of BR in highly trained individuals, there has been no obvious effect of BR, although some individuals seem to be ?responders.? Highly trained endurance athletes benefit from training volume and duration that results in physiological improvements specific to continuous exercise. In this case, the increased endogenous production of NO (Green et al., 2004) coupled with other training adaptations (Joyner and Coyle 2008) may mask the effect of BR.  However, these adaptations do not explain differences in training matched, fit individuals, and little is known about why these responses would differ. Therefore, the specific sport, intensity and duration of exercise may also alter the efficacy of BR. Whether effective in normoxia or not, the decreased PO2 in hypoxia may benefit BR-related performance enhancement in highly trained athletes. BR could potentially ameliorate training intensity and recovery through improved oxygen saturation, economy and blood pressure, which are impacted at altitude. Theoretically, these improvements would benefit both repeated training bouts (such as during a training camp) and competitive performance in hypoxia.	 ?	 ?	 ?	 ? 17	 ?2. Effect of beetroot juice on exercise performance in normoxia and moderate hypoxia 	 ?2.1 Introduction	 ?At altitude, decreases in the barometric pressure ultimately result in less oxygen reaching the tissues (reviewed in Sheel et al., 2010). O2max decreases almost linearly with oxygen saturation (Wagner 2010), limiting intense endurance exercise as a given submaximal workload has approximately the same oxygen cost at altitude as at sea level. Relying on the aerobic energy system to maintain a given workload in hypoxia requires that the individual increase ventilation and cardiac output to improve diffusion and maintain oxygen delivery. Thus, ventilation and cardiac output are higher for a given submaximal workload. The combined effect of increasing cardiopulmonary workloads while lowering maximal aerobic capacity reduces overall training quality, particularly with acute hypoxic exposure in absence of acclimatization. As athletes often train and compete at moderate altitude (i.e., 1500-2500 m), anything that would ameliorate these limiting factors would help to maintain high exercise intensity and performance.  Dietary NO3- lowers blood pressure during exercise recovery in individuals of average fitness (Larsen et al., 2010) and clinical populations (Kenjale et al., 2011). Dietary NO3- also increases mitochondrial efficiency through an improved oxidative phosphorylation ratio (Larsen et al., 2011) in normoxia. Beetroot juice (BR), a supplement with a naturally high concentration of NO3- that may have ergogenic effects for athletes due to subsequent increases in circulating NO3- and NO2- that increase NO availability, has gained popularity with endurance athletes aiming to improve performance; however, normoxic studies in highly trained athletes supplemented with BR indicate that it may be less effective in this population (e.g., Cermak et al., 2012b). Despite evidence that trained athletes may not benefit from BR at sea level, when oxygen is limited in the environment (e.g. at altitude) BR may have even greater physiological effects Muggeridge et al., 2014; Dauncey 2012; Masschelein et al., 2012). Therefore, BR may positively affect trained athletes in hypoxic environments. While Muggeridge et al. (2014) have previously investigated the effect of BR in trained athletes in moderate ?V	 ? 18	 ?hypoxia, a comparison between the efficacy of BR supplementation in normoxia and moderate hypoxia for trained athletes has not been done. This may be useful for understanding under which conditions BR is most effective for athletes. 2.2 Purpose, objectives and hypotheses	 ?The primary purpose of this project is to assess whether an acute dose of BR can improve indices of exercise performance in trained men, and, if so, whether these effects are due to the available [NO3-] in the supplement. There is evidence to suggest that highly trained athletes do not benefit from BR supplementation in normoxia to the same extent as less fit individuals; however, there may be a greater ergogenic effect on performance in hypoxia because it is more difficult to maintain endogenous NO production. To address this question, I examined the efficacy of acute BR supplementation for improved exercise performance in both normoxia and moderate hypoxia in trained male cyclists. Through the course of my thesis, I aimed to address the following four questions by testing the associated hypotheses:  Question 1: Do highly trained athletes experience an ergogenic benefit from BR1 under normoxic conditions?   Question 2: Do highly trained athletes benefit from BR supplementation in hypoxia?  Question 3: Does resting BP decrease following BR supplementation?  Question 4: Does BR supplementation increase fraction of exhaled nitric oxide (FENO)?  Hypothesis 1: BR will have no effect on exercise performance in normoxia (i.e., no change in economy, or TT performance), relative to placebo.  Hypothesis 2: BR will improve indices of exercise performance in hypoxia (i.e., improved economy, increased oxygen saturation, improved TT performance), relative to placebo. 	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?1	 ?For the purpose of this thesis, ?BR? will indicate beetroot juice containing NO3-, while ?PL? will indicate beetroot juice that has been NO3- depleted. The protocol described in this thesis specifically tests whether the NO3- in beetroot juice is effective as an ergogenic aid.	 ?	 ? 19	 ? Hypothesis 3: Acute BR supplementation will decrease resting blood pressure in highly trained men, relative to placebo.  Hypothesis 4: FENO will increase with BR juice supplementation. 2.3 Methods 2.3.1 Participant information Between April and August 2013, 12 trained male cyclists were recruited to participate in this study. Subjects were recruited through poster advertisements at the University of British Columbia (UBC) campus, in cycling shops and through email contact with cycling clubs in Vancouver. Initially, 15 individuals expressed interest in participating; however, two were excluded during screening, as they did not meet all of the inclusion criteria. Of the remaining 13 subjects, one individual did meet the inclusion criteria but withdrew from the study due to personal reasons. Subjects were given $20.00 per visit to the lab ($100.00 total for all five visits). Informed written consent was obtained from all subjects. The UBC clinical research ethics board (CREB) approved this study.   Table 1: Inclusion and exclusion criteria Inclusion Criteria Exclusion Criteria  Males aged 19 (18 for UBC students*) to 40 years Self-reported trained cyclists Maximal oxygen uptake of at least 60 mL?kg-1?min-1 or 5 L?min-1 English speaking  Females History of pulmonary or heart disease Diabetes Smoking Recent (within 2 months) visit to 2500 m or above Inability to communicate in English Note. UBC CREB approved treating 18-year-old UBC students as emancipated for the purpose of this study.  2.3.2 Experimental design A randomized, placebo-controlled, crossover study design was used for this experiment (see Figure 2). The 12 subjects that qualified for the study visited the 	 ? 20	 ?laboratory five times in total. Following an initial screening visit, there were four randomly assigned treatment days: 1) normoxic trial supplemented with BR; 2) normoxic trial supplemented with placebo (PL); 3) hypoxic trial supplemented with BR; and 4) hypoxic trial supplemented with PL. Each visit was separated by at least 96 hours to allow for washout and recovery. All cycling tests were completed on a fully adjustable Velotron cycle ergometer (Racermate Dynafit Pro, Racermate Inc., Seattle WA) to mimic real road cycling as closely as possible. Data collection took place in the Environmental Physiology Laboratory and the Gene, RNA, Informatics and Proteins Laboratory at the University of British Columbia, Vancouver, Canada.  The lab is located approximately 75 m above sea level.    Individual subjects were tested at approximately the same time of day on each visit. Supplementation was administered in a double-blind manner, whereas only the subjects were blinded to the environmental condition.  Informing the researcher of the environmental condition was necessary to allow oxygen saturation to be monitored during the exercise protocol, which was required by CREB. Subjects were given either 70 mL BR or 70 mL PL, and completed the testing protocol in either moderate hypoxia (normobaric hypoxia chamber set to 2500 m) or normoxia. The BR supplement (Beet It Sport, James White Drinks Ltd., Suffolk, UK), which contained beetroot juice and lemon juice had 72 kcal and 0.4 g of NO3- (6.5 mmol NO3-) in 70 mL. The PL product was identical to the BR supplement, except that it was NO3- depleted. This allowed for any differences in the experimental conditions to be explained by the available NO3- content. The supplement was Informed-Sport tested and both the BR and PL were consumed at room temperature. Subjects were asked to record their food intake for the 24-hour period leading up to the initial test and to then replicate this diet as closely as possible over the 24 hours leading up to each subsequent test day. They were also asked to arrive at the lab fully hydrated (i.e., at least 500 mL of water consumed in the preceding hour) but to avoid   alcohol and caffeine consumption for 24 and 6 hours, respectively, before laboratory visits.  As well, subjects were asked not to use anti-bacterial mouthwashes or chew anti-bacterial gum for the duration of the study. Other than the treatment, subjects did not eat or drink anything during the testing with the exception of water, which was permitted ad 	 ? 21	 ?libitum between supplementation and exercise testing. Finally, subjects did not exercise on test days and remained in the laboratory between supplementation and exercise testing.   Figure 2: General study overview. Subjects came to the laboratory on five separate occasions. The first visit included a maximal exercise test and familiarization with the exercise protocol to be completed on subsequent days. During data collection days, subjects underwent one of four protocols: supplementation with either beetroot juice (BR) in hypoxia or normoxia, or supplementation with nitrate-depleted beetroot juice (PL) in either normoxia or hypoxia (see text for details).  2.3.2.1 Visit 1: Fitness screening 	 ?The first visit consisted of anthropometric measurements, baseline fitness testing and familiarization with the lab, the equipment to be used on test days, and the experimental day protocol (see Appendix D for subject information sheets). Subjects were weighed and measured. A venous blood sample was taken to measure baseline plasma [NO3-] and [NO2-], and a finger prick sample was drawn into a capillary tube to determine hematocrit using a CMH30 Micro Hematocrit Centrifuge (UNICO, Dayton NJ, USA). To determine whether prospective subjects met the inclusion criteria for fitness, each individual completed an incremental O2max test to volitional exhaustion. A mouthpiece was fitted to each subject?s face fixed with a two-way, non-rebreathing valve. Subjects inhaled room air and exhaled to a metabolic cart via a breathing tube. Subjects completed a self-selected, five-minute warm-up on the Velotron cycle ergometer. The test started immediately afterwards at a work rate of 0 W. The work rate increased by 1 W every 2 seconds. O2max was met when the subject met at least three of the following criteria: 1) a plateau in oxygen uptake despite an increase in work rate; 2) a respiratory ?V?VBaseline	 ?VO2max BR	 ?+	 ?hypoxia Time	 ?trial BR	 ?+	 ?normoxia Time	 ?trial PL	 ?+	 ?hypoxia Time	 ?trial PL	 ?+	 ?normoxia Time	 ?trial Randomized,	 ?crossover	 ? SCREENING	 ?DAY	 ?(Visit	 ?1) EXPERIMENTAL	 ?DAYS	 ?(Visits	 ?2-??5) 	 ? 22	 ?exchange ratio >1.15; 3) heart rate > 90% of the age predicted max (220 bpm ? age in years); 4) volitional exhaustion. The subject then completed a self-selected cool down. For those subjects who achieved a O2max value ?60 mL?kg-1?min-1 (or 5 L?min-1) this ramp test was also used to determine their 50% maximal power outputs (in Watts) for subsequent test days. Each subject?s 50% power output was calculated from power output at O2max. After a rest period, subjects completed the TT protocol in normoxia (described in detail below), used for subsequent testing days, to familiarize them to the ergometer?s gearing and the intensity and duration of the TT protocol.   2.3.2.2 Visits 2, 3, 4, 5: Data collection	 ?On arrival to the lab, resting blood pressure (BP) was assessed manually with a sphygmomanometer and a stethoscope. Subjects sat quietly for five-minutes and kept both feet on the floor during BP assessment. BP was measured in duplicate, and the second measurement was used for analysis. Fraction of expired nitric oxide (FENO) was measured following the American Thoracic Society and European Respiratory Society guidelines for measurement of exhaled lower respiratory nitric oxide using offline measurements (ATS and ERS 2005). While in a seated position, subjects held the NIOX MINO handheld electrochemical analyzer (Aerocrine AB, Solna, Sweden). Subjects exhaled completely, inhaled NO-free air through the mouthpiece until reaching vital capacity, and immediately exhaled for 10 seconds at a rate of 50 mL?s-1 to complete the test. FENO was measured once. Immediately following these tests, subjects drank 70 mL of either BR supplement or PL (distributed using a double blind protocol). Regardless of treatment subjects waited in the laboratory for two hours, to allow time for any increase in plasma [NO3-] and [NO2-]. After two hours, post-supplementation blood pressure and FENO were assessed and a second venous blood sample was taken.  Following post- supplementation tests, subjects were fitted with a heart rate monitor (Polar T31 Transmitter, Polar; Kempele, Finland), an earlobe pulse oximeter (Avant 9600, Nonin Instruments, Plymouth, MN), and a facemask (Hans Rudolph, Oro-Nasal 7450 V2 Mask, Shawnee, KS) connected to a two-way non-rebreathing valve ?V?V	 ? 23	 ?(2700 T-shape, Hans Rudolph, Shawnee, KS). The valve was attached on one side to a normobaric hypoxia chamber (Colorado Altitude Training; Louisville, CO) with a breathing tube, and to a metabolic cart (Parvo Medics; Sandy, UT) on the other side with another breathing tube (see Figure 3).    Fig 3: Breathing set-up. Subjects breathed either normoxic or hypoxic air from the normobaric hypoxia chamber through a breathing tube, and exhaled through a second tube to a metabolic cart.  Prior to warming up on the cycle ergometer, emission and detection fibre optic probes connected to a near infrared spectroscopy (NIRS) system (NIRO-200 Oxygenation Monitor, Hamamatsu, Japan) were taped to the subject?s upper thigh (over the vastus lateralis) to assess tissue oxygenation index (TOI). The probes were covered with a tensor bandage, which lay under the subjects? cycling shorts. The site of probe placement was marked on the skin in permanent marker, so probe position could be replicated for subsequent test days. Muscle oxygenation data was collected during the steady state and TT portions of the cycling protocol. Subjects were seated on the cycle ergometer, facing a computer screen, breathing either ~21% or ~16% O2 from the normobaric hypoxia chamber. A 15-minute self-selected warm-up on the cycle ergometer was then completed followed by 15 minutes at 50% of each subject?s specific maximal power output (as pre-determined by their initial O2max test). During the steady state portion of the test, power output was fixed, and subjects were asked to maintain a seated position and a consistent cycling cadence. ?V	 ? 24	 ?Metabolic data was measured over the final five minutes of this task. This data was used to assess exercise economy (mL O2?min-1?W-1), the main outcome variable for the steady state portion of the test. Subjects were not given any feedback regarding gearing or cadence during the warm-up to prevent subjective comparison with future and past testing days. Following the steady state exercise, subjects completed a 10-km TT on a flat and straight course (RacerMate Interactive 3D Software, Seattle, WA) (see Figure 4 and Table 3). During the TT, subjects were able to change gears and position if desired. Only feedback on distance covered and cadence was provided to them via the computer screen. Metabolic data was collected for the duration of the TT. The main outcome variable for the TT was mean power output relative to body weight (W?kg-1).  Following completion of testing protocols, subjects were asked whether they thought they had experienced hypoxia or normoxia and whether they consumed BR or PL. This was used to assess the efficacy of the single- and double- blinding. See Appendix C for a more detailed explanation of the normoxic and hypoxic breathing protocol, as well as metabolic calculations.                         	 ? 25	 ?Table 2: Summary of procedures for all subjects on laboratory visits.  Appointment 1 Appointments 2, 3, 4, 5  -Anthropometric measures (height, weight)  -Hematocrit measurement  -Incremental maximal oxygen uptake test  ( O2max)  -BP and FENO  -Supplemented with either 70 mL BR or PL   -2 hours post-supplementation, second BP and FENO  -15 minute self-selected warm up, breathing either normoxic (~21% O2) or hypoxic air (~16% O2), followed by 15 minutes at 50% max power (HR, SpO2, TOI, O2, power output)  -10-km time trial (SpO2, TOI, O2, power output)  Note. NO3-=nitrate; NO2-=nitrite; O2max =maximal oxygen uptake; FENO=fraction of expired nitric oxide; HR=heart rate; BP=blood pressure; SpO2=pulse oxygen saturation; O2max =oxygen uptake; TOI=tissue oxygenation index.    2.3.3 Statistical analysis A power calculation (G*Power 3.1, Heinrich Heine, Universit?t D?sseldorf) indicated that the required sample size for this study was 12 subjects. This was based on submaximal cycling economy data from Bailey et al. (2009). This study showed that the mean cycling economy of the placebo condition was 11.6 (SD = 0.9) and the mean of the nitrate-supplementation condition was 10.8 (SD = 0.8). This power calculation was based on an alpha level of 0.05 and a power of 0.84. Descriptive analyses of all variables are presented as mean (SD). A two-way repeated measures ANOVA was used to analyze differences between the four conditions for BP and FENO variables. The independent variables for these tests were time (pre- and post- supplementation) and treatment (PL or BR). Dependent variables were systolic BP, diastolic BP and FENO. Paired samples t-tests were used to compare the average difference in FENO between pre- and post- supplementation with BR or PL. For these analyses, averages of the two BR and two PL test days were used. ?V ?V ?V?V ?V	 ? 26	 ?A two-way repeated-measures ANOVA was used to analyze differences between the four conditions for dependent variables relating to steady state and TT performance. The independent variables for these tests were condition (hypoxia, normoxia) and supplement treatment (BR, PL). For the steady state portion of the protocol, dependent variables were cycling economy (mL O2?min-1?W-1), SpO2, TOI, HR, BP, and VE. In the TT, dependent variables were time to complete 10-km, mean power output per kg, and mean O2. For all tests, the alpha-level was set at p = 0.05.   Fig. 4: Timeline of data collection (sessions 2-5). After pre-supplementation blood sampling and tests for expired nitric oxide (FENO) and blood pressure (BP)(1), subjects consumed either 70 mL beetroot juice (BR) or placebo (PL)(2). Following a two-hour break, blood sampling and FENO and BP tests were completed again (3). Subjects were then exposed to either normoxia or hypoxia, and completed a cycling exercise protocol consisting of a 15-minute self-selected warm-up (4), 15 minutes at 50% maximum power output (5) and a 10-km time trial. Each session concluded with a cool-down. 	 ?2.4 Results  2.4.1 Subject characteristics Twelve healthy, trained male cyclists and triathletes volunteered to participate in this study. Subject anthropometric data is presented in Table 4.       ?VArrival 1 2 2 hr break 3 4 0 6 5 7 Hypoxia or normoxia 15 150 195 Time in lab (min) 	 ? 27	 ?  Table 3: Subject anthropometric, fitness, and power data. Statistic Age (yrs) Height (cm) Weight (kg) O2max (ml?kg-1?min-1) 50% max power (W) Hematocrit (%) Mean  29.3 181 75.9 67.5 211 45 SD  5.1 5.2 6.5 5.8 13.1 3 Min  21 173.5 65.4 60.9 190 38 Max 40 187.5 86.8 81.2 230 50 Note. n = 12, except for hematocrit where n = 10. O2max, maximal oxygen consumption (mL?kg-1?min-1).  2.4.2 Hypoxic and normoxic exposures The PO2 did not differ between PL and BR supplementation days in hypoxia (117 [1] and 118 [1] mmHg, respectively. t[11] = -1.659; p = 0.125), or normoxia (155 [1] and 155 [2], respectively. t[11] = -0.312; p = 0.761). The average PO2 for hypoxic exposures corresponded to an altitude of 2440 m (~8000 feet). Subjects correctly identified the environmental condition on 56% of trials, while 21% of assumptions were incorrect and 23% did not know.  2.4.3 Resting blood pressure and FENO Resting SBP and DBP were similar before and after BR and PL supplementation (see Figure 5). Mean FENO was similar before (25 [11] vs. 23 [8] ppb) supplementation with either BR or PL. There was a significant interaction effect for time x treatment, and follow up paired samples t-tests indicate FENO post-BR was significantly greater than post-PL (28 [12] vs. 22 [9] ppb) (see Tables 4 and 5). Subjects correctly identified the supplement 8% of the time and incorrectly identified the supplement 11% of the time (81% did not know.) Descriptive data and average difference data for BP and FENO variables are reported in Tables 5 and 6.     ?V?V	 ? 28	 ?  Fig 5: Systolic and diastolic blood pressure (mmHg) pre- and post-supplementation with either placebo (PL) or beetroot juice (BR). Error bars represent standard deviation.    Table 4: Summary data for blood pressure and fraction of expired nitric oxide measurements pre- and post- supplementation. Variable Time Supplement N Mean [SD] Paired samples t-test and p-value SBP  (mmHg) Pre-supplement BR PL 12 12 116 [7] 116 [7] t[11]=0.114;  p=0.911 DBP (mmHg) Pre-supplement BR PL 12 12 73 [6] 74 [8] t[11]=-0.435;  p=0.672 SBP (mmHg) Post-supplement BR PL 12 12 115 [7] 116 [8] t[11]=-0.543;  p=0.598 DBP (mmHg) Post-supplement BR PL 12 12 73 [6] 75 [7] t[11]=-0.758;  p=0.464 FENO (ppb) Pre-supplement BR PL 12 12 25 [11] 23 [9] t[11]=1.484;  p=0.166 FENO (ppb) Post-supplement BR PL 12 12 28 [12]* 22 [9] t[11]=5.016;  p<0.001 Note: BR, beetroot juice; PL, placebo; SBP, systolic blood pressure; DBP, diastolic blood pressure; FENO, fraction of exhaled nitric oxide. (*) Indicates a significant difference from the PL treatment.                	   	 	 ? 29	 ?2.4.4 Steady-state cycling Variables measured during the 50% power output trial are presented in Table 7. Relative to normoxia, hypoxic exposure increased VE and HR and decreased SpO2 and TOI (p < 0.05 for all variables). There was no difference in O2 or exercise economy (p > 0.05 for both variables) between normoxic and hypoxic conditions. In normoxia, there was no effect of BR on VE, HR, O2, or exercise economy between BR and PL trials. In hypoxia, there was no effect of BR on VE, HR, SpO2, TOI, O2, or economy (p > 0.05 for all variables.) See Figure 7 below and Figures 8-11 in Appendix E.  Fig 6: Economy (mL O2?min-1?W-1) during steady state cycling at 50% maximum power output in normoxia or hypoxia when supplemented with either placebo (PL) or beetroot juice (BR). Error bars represent standard deviation.   2.4.5 10-km time trial Relative to normoxia, TT performance was significantly decreased in hypoxia. Time to complete the 10-km TT and increased, and mean power output decreased in hypoxia relative to normoxia (p < 0.05 for both variables). Mean O2 and SpO2 were also significantly lower in hypoxia (see Table 8). TOI was similar between both conditions (see Table 8).  In normoxia, there was no improvement in performance with BR. There was no difference in time to complete the 10-km TT, mean power output, or mean O2 over 10 km (p > 0.05 in all variables). In hypoxia, there was also no improvement in performance with BR. Mean O2 over 10 km, time to complete the 10-km TT, mean power output, ?V?V ?V	  	?V ?V?V	 ? 30	 ?SpO2 and TOI did not differ between BR and PL trials. See Table 8 for summary statistics of variables measured during the 10-km TT. See also Figure 8 below and 14-18 in Appendix F.  Fig 7: Mean power output (W?kg-1) during the 10-km time trial in either normoxia or hypoxia when supplemented with placebo (PL) or beetroot juice (BR). Error bars represent standard deviation.         Mean power (W? kg-1 )Normoxia Hypoxia012345PLBRP<0.001	 ? 31	 ?Table 5: Summary statistics for systolic and diastolic blood pressures and fraction of expired nitric oxide pre- and post- supplementation with BR or PL. Variable n Pre-PL Pre-BR Post-PL Post-BR ANOVA Results Time Treatment Interaction SBP (mmHg) 12 116 [7] 116 [7] 116 [8] 115 [7] F(1,11)=0.178 P=0.681 F(1,11)=0.077 P=0.786 F(1,11)=0.843 P=0.378 DBP (mmHg) 12 74 [8] 73 [6] 75 [7] 73 [6] F(1,11)=0.254 P=0.625 F(1,11)=0.460 P=0.512 F(1,11)=0.176 P=0.683 FENO (ppb)  12 23 [8] 25 [11] 22 [9] 28 [12] F(1,11)=0.946 P=0.352 F(1,11)=11.219 P=0.006* F(1,11)=15.621 P=0.002* Note. BR, beetroot juice; PL, placebo; SBP, systolic blood pressure; DBP, diastolic blood pressure; FENO, fraction of expired nitric oxide. Data presented as mean [SD].    	 ? 32	 ?Table 6: Summary statistics for physiological responses to cycling in normoxia and hypoxia at 50% of maximum power following supplementation with BR or PL Variable n NPL NBR HPL HBR ANOVA Results Treatment Condition Interaction ?V O2 (mL?kg-1?min-1) 12 39.3 [3.6] 39.7 [3.8] 38.5 [5] 39.1 [3.6] F(1,11)=0.412; p=0.534 F(1,11)=0.785; p=0.395 F(1,11)=0.012 p=0.914 Economy  (mL O2?min-1?W-1) 12 14.2 [1.1] 14.3 [1.2] 13.9 [1.3] 14.1 [1.2] F(1,11)=0.248 p=0.682 F(1,11)=0.670 p=0.430 F(1,11)=0.057 p=0.816 VE (L?min-1) 12 80.7 [17] 76.5 [10] 88.1 [9.9] 91.2 [13.9] F(1,11)=0.097 p=0.761 F(1,11)=31.272 p<0.001* F(1,11)=1.949 p=0.190 HR (bpm) 12 150 [13] 149 [11] 156 [9] 156 [12] F(1,11)=0.218 p=0.650 F(1,11)=34.726 p<0.001* F(1,11)=0.061 p=0.810 SpO2 (%) 12 98 [1] 98 [2] 87 [4] 88 [3] F(1,11)=2.926 p=0.115 F(1,11)=156.708 p<0.001* F(1,11)=1.347 p=0.270 TOI (%) 7 51 [12] 52 [13] 45 [12] 46 [10] F(1,6)=0.161 p=0.702 F(1,6)=13.785 p=0.01* F(1,6)=0.023 p=0.884 Note. BR, beetroot juice; PL, placebo; ?V O2 (mL?kg-1?min-1), oxygen uptake; VE, ventilation; HR, heart rate; SpO2, pulse oxygen saturation; TOI, tissue oxygenation index. Data presented as mean [SD].  Table 7: Summary statistics for physiological responses to a 10-km cycling time trial in normoxia and hypoxia following supplementation with BR or PL. Variable n NPL NBR HPL HBR ANOVA Results Treatment Condition Interaction Mean ?V O2 (mL?kg-1?min-1) 12 50.5 [5.5] 51.2 [5.3] 43.3 [7.4] 44.4 [5.4] F(1,11)=0.681 p=0.427 F(1,11)=29.513 p<0.001* F(1,11)=0.009 p=0.925 Mean power output (W?kg-1) 12 3.8 [0.4] 3.7 [0.4] 3.2 [0.3] 3.2 [0.3] F(1,11)=0.379 p=0.550 F(1,11)=161.971 p<0.001* F(1,11)=2.505 p=0.142 Time (s) 12 950 [47] 959 [53] 1023 [47] 1015 [51] F(1,11)=0.006 p=0.939 F(1,11)=143.784 p<0.001* F(1,11)=2.385 p=0.151 SpO2 (%) 12 97 [2] 97 [3] 87 [4] 87 [4] F(1,11)=0.363 p=0.559 F(1,11)=365.943 p<0.001* F(1,11)=0.156 p=0.692 TOI (%) 7 48 [12] 49 [13] 46 [10] 45 [11] F(1,6)=0.013 p=0.913 F(1,6)=5.848 p=0.06 F(1,6)=0.177 p=0.692 Note. BR, beetroot juice; PL, placebo; Mean ?V O2, mean oxygen uptake over 10-km; SpO2, pulse oxygen saturation; TOI, tissue oxygenation index. Data presented as mean [SD].	 ? 33	 ?2.5 Discussion	 ? 2.5.1 Layout of discussion The purpose of this study was to assess the effects of BR supplementation on exercise performance in trained male cyclists in normoxia and moderate hypoxia. The discussion is divided into three parts: 1) the effects of BR on pre- and post- supplementation variables; 2) the effects of BR on exercise economy; and 3) the effects of BR on 10-km TT performance.   2.5.2 Pre- and post-supplementation variables The supplement used in this study is sold as an ergogenic aid, and was selected as appropriate for practical application to the target athlete group as it is commercially available and inexpensive. Furthermore, the NO3- content of the supplement that we used  (400 mg) falls within the suggested ?safe? limits of NO3- intake of 300-500 mg (Lundberg et al., 2011a). Despite the suggested benefits of NO3-, little is known of the long-term effects and safe amounts for consistent use. In the past, there have been concerns regarding high concentrations of NO3- and NO2- in drinking water. If NO2- binds to hemoglobin and oxidizes the heme group to its ferric state (Fe3+), the reaction forms methemoglobin, which cannot bind with O2 (Lundberg et al., 2008). Infants under six months have a smaller amount of the enzyme that converts methemoglobin back to hemoglobin, making them vulnerable to methemoglobinemia (?blue baby syndrome?) (Avery 1999). Methemoglobinemia is very unlikely in adults without underlying pathology, but it remains a worthwhile consideration. Consumption of meat products preserved with NO2- may be linked to cancer; however, epidemiological studies have not demonstrated conclusive evidence of this belief (Mensinga et al., 2003). Nonetheless, uncontrolled intake of NO3- supplements could result in NO3- toxicity (Lundberg et al., 2011a). Anecdotally, some people find that foods containing dietary NO3- or NO2- trigger headaches, and migraines. To date, there have been no reported headaches in BR studies, although some individuals may be susceptible to this effect. This would be particularly relevant in hypoxia, where headache is a symptom of acute mountain 	 ? 34	 ?sickness (Hackett and Roach 2001). However, it is obvious from other studies that continuous supplementation with BR over multiple days (e.g., Vanhatalo et al., 2010) or a larger acute dose (e.g., Wylie et al., 2013a) causes greater increases in plasma NO3- and NO2- than a single concentrated dose (e.g., Wylie et al., 2013a).  Our study is the first to measure the effect of BR on FENO, and our results show that an acute dose of ~6.5 mmol [NO3-] in 70 mL BR is sufficient to increase FENO. Previously, a meal rich in NO3- has been demonstrated to increase FENO in healthy individuals (Olin et al., 2001).  Dietary NO3- and NO2- that are not reduced in the acidic stomach may be absorbed from the intestine to the blood. As blood passes through the lungs, it is possible that some NO3- or NO2- carried in the blood is reduced to NO.   2.5.3 Blood pressure  Supplementation with BR did not decrease resting BP in this study. BR supplementation has demonstrated a small but consistent drop in systolic blood pressure (Siervo et al., 2013), and a recent study by Wylie et al. (2013a) indicated that systolic BP significantly decreased following acute supplementation with 70 mL, 140 mL or 280 mL of BR containing 4.2, 8.4 and 16.8 mmol NO3-, respectively. Therefore, the dose of BR used in our study should have been sufficient to decrease BP; however, these authors also indicated that the peak reduction in BP following the smallest dose of BR was at 4 hours post-supplementation. In the present study, subjects? BP was assessed two hours post-supplementation, which may not have allowed enough time for BP to decrease. Metadata presented by Siervo et al., (2013) included data from individuals with health comorbidities, where BP may be more responsive to NO3- or BR supplementation. Subjects in our study had fairly low BP and perhaps BR would reduce BP more in individuals with higher baseline BP. It is also possible that physiological arousal was high immediately prior to completing the TT protocol, which could counteract any lowering effects of the BR. It is worth noting that this study was not specifically powered to detect a change in blood pressure.  	 ? 35	 ?2.5.4 Exercise economy In our study, economy was similar between BR and PL trials in normoxic and hypoxic steady-state trials. Economy also did not differ significantly between hypoxia and normoxia. Any improvement in economy would likely be indicative of alterations in oxidative phosphorylation, as a given amount of work should have a consistent energy cost (Jones n.d). Our data is inconsistent with previous evidence demonstrating measurable improvements in exercise economy with BR supplementation. Previous studies investigating the effect of BR on economy reported improvements with BR supplementation in normoxia relative to PL. Lansley et al. (2011b) concluded that economy was significantly improved following BR (0.5 L (~5 mmol NO3-) per day for 15 days prior to exercise) during both moderate- and severe-intensity treadmill exercise. Using a similar six-day loading protocol to Lansley et al. (2011b), Bailey et al. (2009) also demonstrated lowered oxygen cost at the end of exercise for moderate intensity cycling; however, the provided dose of NO3- in the BR supplement was greater in these studies than the supplement used in the present study. It seems promising that a greater acute NO3- dose will result in greater benefit to performance (Hoon et al., 2013a), although smaller doses of BR over a few days would likely be more palatable to athletes.  One of the discrepancies between this study and those previously published is the fitness of subjects. The majority of studies have used recreationally active subjects, while our study used well-trained subjects; however, Cermak et al. (2012a) demonstrated a lower O2 in trained cyclists during 30-minute steady state cycling at 45% and 65% of maximum power output, when supplementing with 140 mL BR per day (~8 mmol NO3-) over six days. The daily dose in that study was greater than the acute dose provided in our study. Data from Cermak et al. (2012a) indicated that BR may be effective in trained athletes, but athletes may require a larger dose than untrained individuals to see a benefit in performance. Furthermore, highly trained athletes routinely cause sufficient shear to their vessels to up-regulate NO production through the endogenous NOS system. Training adaptations specifically affecting NO production may mask the effect of a small, acute, dose of BR on economy in the trained athletes in this study. Studies showing an improvement in economy indicate that loading protocols ?V	 ? 36	 ?are a viable option to improve economy; however, large acute doses may be more practical for athletes. Different loading and acute supplementation protocols, as well as fitness, are therefore important considerations for athletes competing in normoxia. The NO3- dose per kg of body weight could also be an important mediator of the effects. The average weight of the subjects in our study was 75.9 [6.5], ranging from 65-87 kg; however, each individual was provided with the same dose of BR. It is possible that the NO3- dose per kg was not sufficient to cause a measurable effect on economy in the heavier subjects. However, there was no change in economy in our study and no correlation between dose by weight and economy.   The acute dose of BR used in this study was not expected to have an effect in normoxia in our subject population due to their baseline fitness (i.e. subjects were well trained). While the NOS system is upregulated in hypoxia (Casey and Joyner 2012), the lack of available oxygen inhibits oxidation of L-arginine, which we hypothesized would increase the efficacy of BR; however, steady-state economy was similar between BR and PL in hypoxia. The NO3- ? NO2- ? NO system is potentiated by hypoxic environments (Dauncey 2012), and the conversion of NO3- to NO2- is maximized as oxygen saturation approaches 50% (Lundberg et al., 2008). Furthermore, this system is primarily anaerobic. Hypoxic exposure affects aerobic performance to a greater extent in fit individuals compared to individuals of lower baseline fitness (Fulco 1998). If highly trained individuals are affected more by hypoxia (and if they become more hypoxemic than less fit individuals (Dempsey and Wagner 1999); it follows that they will have less oxygen available for their NOS systems, making the anaerobic NO pathway particularly useful to fit individuals in hypoxia. Our normobaric hypoxic exposure was equivalent to moderate altitude (~2440 m), which may not be sufficient to negatively impact NOS production in the trained subjects. Another possibility is that the BR dose we used was not great enough to enhance the effects of endogenous NO production to a degree that would be measurable within the normal day-to-day variation of metabolic data. Our results disagree with those of Muggeridge et al. (2014), which indicated a measurable benefit to performance in trained athletes supplemented with BR in moderate hypoxia. Muggeridge et al. (2014) had a very similar protocol to our study: trained cyclists completed a 15-minute steady-state cycling bout at 60% of hypoxic 	 ? 37	 ?maximum power output at a simulated 2500 m. The authors concluded that O2 was lower in the BR trial compared to PL (2542 [114] mL.min-1 vs. 2727 [85] ml?min-1 p = 0.049). The acute BR supplementation protocol in this study was similar to ours, although it had a lower NO3- content (70 mL containing ~5 mmol NO3-). Muggeridge et al. (2013b) provided the BR supplement three hours before performance testing as opposed to our two hour interval, which may not have allowed enough time for plasma [NO3-] and [NO2-] to peak, and reduced the likelihood of BR benefitting performance. Muggeridge et al. (2013b) also based their hypoxic steady state intensity on a hypoxic  O2max test. In our study, 50% of maximum power output was based on a normoxic     O2max test. It is likely that in our study, 50% power in normoxia was equivalent to a greater percentage of maximal workload in hypoxia, making it more difficult to maintain metabolic steady state. However, while the exercise intensity had a greater physiological cost (e.g., VE and HR were higher) in hypoxia compared to normoxia, hypoxic exposure for both BR and PL trials was consistent, allowing for direct comparison between hypoxic steady state trials. Furthermore, measured economy and O2 did not differ between normoxic and hypoxic steady state trials in our study. While subjects may have been slightly less economical in hypoxia, it was not obvious from our collected metabolic data that the chosen intensity was too high to maintain steady state, despite subjects working harder in hypoxic than in normoxic trials.   The HR and VE were significantly increased and SpO2 and TOI significantly decreased during the hypoxic PL economy trial compared to the normoxic PL trial in our study; however, these variables were unchanged between BR and PL trials in both normoxia and hypoxia. Previously, Masschelein et al. (2012), demonstrated improvement in SpO2 as well as TOI in the vastus lateralis during steady state cycling at 45% maximum power after supplementing with a BR dose of 0.07 mmol NO3-?kg-1?day-1 for six days prior to testing (equating to ~5.25 mmol NO3- in a 75 kg individual). In that study, hypoxic exercise trials were completed at a simulated altitude of 5000 m, making the exposure more severe than ours, and mean SpO2 during steady-state exercise was below 70%. On average, SpO2 during hypoxic steady-state cycling in our study was 88% with BR and 87% with PL; given that we did not find a difference in saturation between ?V?V?V ?V	 ? 38	 ?the supplement trials, it is possible that there needs to be a greater degree of hypoxemia for BR to have an effect on blood oxygen saturation. Similar to our study, Muggeridge et al. (2013b) demonstrated no difference in steady-state SpO2 between BR and PL trials at a simulated altitude of 2500 m. At an oxygen saturation of ~88%, oxygen concentration remains on the relatively flat portion of the oxygen dissociation curve. It is possible that if BR attenuated a decrease in saturation through NO availability, it would be more obvious at higher altitudes, because the conversion of NO2- to NO is potentiated as oxygen tension falls and the oxygen dissociation curve becomes much steeper (i.e., small effects would cause a greater shift in SpO2). If this were correct, the efficacy of BR supplementation would correlate with severity of hypoxic exposure.   2.5.5 10-km TT performance Acute BR supplementation did not improve 10-km TT performance relative to PL in either normoxia or hypoxia in our study: there was not a significant difference between time to complete the TT, mean power output or mean O2. TOI and SpO2 did not differ between BR and PL supplementation. As expected, hypoxia impaired TT performance:  SpO2 and mean O2 were significantly lower, time to complete 10-km significantly increased, and mean power output was decreased by ~17% in hypoxia relative to normoxia. Data from Muggeridge et al. (2013b) indicated an improvement in 16.1-km TT performance following acute BR supplementation at a simulated altitude of 2500 m (70 mL; ~5 mmol NO3-); however, as discussed earlier, these authors tested their subjects three hours post-supplementation, potentially allowing more time for NO3- and NO3- to peak. Previous studies testing trained cyclists suggested that some individuals are ?responders? to BR supplementation. Christensen et al., (2012) supplemented with 500 mL (0.5 g NO3-, ~8 mmol NO3-) BR per day for six days and had subjects complete a two-hour cycling warm-up consisting of moderate- to high-intensity blocks, followed by a 400 kcal TT. Although there was no significant improvement with BR, these authors identified two responders ( O2max of 71 and 74 ml?kg-1?min-1) as individuals who had a ?V?V?V	 ? 39	 ?2.5% and 8% improvement, respectively, in the 400 kcal TT. These individuals did not differ from the rest of the subjects in baseline plasma [NO3-]. Wilkerson et al. (2012) also did not report an overall significant improvement in trained cyclists over a 50-mile TT with an acute 500 mL dose of BR containing 6.2 mmol NO3-; however, six of eight subjects demonstrated a performance improvement, and O2 tended to be lower, resulting in an improved economy.   Unlike the two studies of highly trained cyclists discussed above (Christensen et al., 2012; Wilkerson et al., 2012), individual subjects were not identified as responders in our study. Subjects were not tested repeatedly in each environmental condition and we therefore could not conclude whether any differences in performance between BR and PL were due to chance or the effect of supplementation, on an individual basis (i.e. when the overall result is no effect, some subjects will inevitably demonstrate an effect (?responders?) and some subjects will not (?non-responders?), as data will be dispersed around the mean). Mean O2max (67.5 [5.8] ml?kg-1?min-1) in our study was slightly higher than the average in the Wilkerson et al. (2012) (63 [8] ml?kg-1?min-1). The NO3- dose in the BR supplement was similar between the two studies, but differences in results are unlikely due to a difference in fitness. However, subjects tested by Christensen et al. (2012) had a mean O2max of 72.1 [4.5] ml?kg-1?min-1, indicating that while fitness likely affects the efficacy of BR, it may not fully explain the discrepancy between our study and previous research. If BR does have an effect, the most likely reason for this discrepancy could be a combination of subject weight and acute standardized dosage. However, on average, our subjects were similar to subjects in these studies (75.9 [6.5] kg in our study vs. 69 [8] kg (Christensen et al., 2012) and 79 [9] kg (Wilkerson et al., 2012), but were provided with a small, acute dose of NO3- (i.e., 6.5 mmol or ~0.09 mmol?kg-1). While this dose was nearly equivalent by weight to the Wilkerson et al., (2012) study (0.08 mmol?kg-1) where most subjects improved with BR, Christensen et al., (2012) provided subjects with a dose of ~0.11 mmol?kg-1 over six days and identified only two subjects as benefitting from BR. The relative dose-by-weight of the subjects with the greatest improvement with BR in these studies are unknown. Cycling protocols in these studies were also different from the present study, with both protocols having a longer duration warm-up and TT relative to our study. As ?V?V ?V	 ? 40	 ?mean power output remained high in these other studies, it is possible that the effects of BR may be more evident in longer-duration exercise as [NO3-] and [NO2-] peak; however, it should be noted that Cermak et al. (2012b) reported that acute BR supplementation with ~8.7 mmol NO3- did not improve performance of a 1073 [21] kJ (~1-hour) TT. Average power output during that trial was reasonably high (275 [7] W with BR vs. 278 [7] W with PL), corresponding to ~70% maximal power output. Finding a balance between dose-response and exercise duration and intensity should be a primary goal for individual athletes wishing to improve performance with BR. Improvement of shorter TT distances has also been reported with BR supplementation. Cermak et al. (2012a) demonstrated an improvement in a 10-km TT in trained cyclists supplemented with 140 mL BR (~8 mmol NO3-) over 6 days; however, their study involved a supplement loading protocol and a longer warm-up (allowing more time for plasma NO2- or NO3- to peak) than our study, both factors that may play a role in enhancing BR-related performance gains.  Overall, results from studies in the literature indicated that the effect of BR is smaller in trained athletes than in untrained individuals, and that the effect is nearly undetectable for TT performance (e.g., G = 0.11, 95% CI = -0.16, 0.37) (Hoon et al., 2013b). While these were expected outcomes in our study in normoxia, our hypothesis that any effect of BR would be more obvious in hypoxia was not supported by our data, in contrast to results from Muggeridge et al. (2013b). As the most directly comparable study, these authors demonstrated a significant performance improvement in time to complete a 16.1-km TT, with nine subjects and a calculated effect size of D = 0.67. Both our study and Muggeridge et al. (2013b) had small sample sizes (twelve and nine subjects, respectively). Therefore any differences in our conclusions are most likely the result of a lack of statistical power due to small sample size resulting in high sampling variability. Considering the conservative effect size of BR (G = 0.11) suggested by Hoon et al. (2013b), and assuming an alpha level of 0.05, with the 12 subjects tested in this study, we would have a 6% chance of detecting an effect with BR. Using the same assumptions, Muggeridge et al. (2013b) would have a 5% chance of finding the effect of BR that they reported. If we assume that BR would be more effective in hypoxia than in normoxia, and assume the upper limit effect size calculated by Hoon et al. (2013b), G = 	 ? 41	 ?0.37, we would have a 22% chance of demonstrating an effect of BR, and Muggeridge et al. (2013b) would have a 16% chance. If we test the effect size suggested by Muggeridge et al., (2014) in hypoxia, D = 0.67, these authors would have a 42% chance of finding their original result in a follow-up experiment (with their sample size of nine subjects). Our study would have a 56% chance of detecting a difference with BR with the same assumptions (n=12). Therefore, it is likely that the effect of BR falls somewhere between the two studies, although the main difference is likely sampling variability. With a calculated Cohen?s D of 0.18 in our study, and of 0.67 in the study by Muggeridge et al. (2013b), the weighted mean of the effect of BR in hypoxia is 0.39. For 80% power, we would require 43 subjects to see an effect of BR. While the data in our study do not directly indicate that BR would be effective for improving TT performance, the results are in the direction of our hypothesis. Thus, in the future studies investigating BR for TT performance in hypoxia would need to increase sample size, or change other variables (e.g., increase altitude, increase supplement) to increase statistical power. While the sample size could be increased in order to show a ?statistically significant finding,? it is also worth considering that the effect appears to be small and highly variable. In this regard, BR is unlikely to negatively affect performance, but it may not be worthwhile for athletes to consume BR when competing in hypoxia in lieu of other methods to legally and safely enhance performance, whether currently known or yet to be discovered.   2.5.6 Summary  The primary outcome of this study was that cycling performance was not improved following supplementation with BR relative to PL, although performance was negatively impacted by hypoxic exposure. There were no differences in economy or time trial performance between BR and PL trials. Resting blood pressure was also similar pre- and post- supplementation with BR or PL trials and FENO did not increase post-BR supplemention, indicating that the supplement was not sufficient to increase NO production in the body.	 ?  	 ? 42	 ?3. Conclusion  BR supplementation has become popular with athletes, although the few studies investigating highly trained individuals in normoxia indicate that its efficacy may be limited in this population. Aerobic capacity is decreased in hypoxia and highly trained athletes experience greater decrements in endurance performance than untrained individuals in hypoxia. Previous research has indicated that even if athletes do not benefit from BR in normoxia, the supplements may be ergogenic at altitude. The proposed mechanisms of action behind BR supplementation may be particularly effective in hypoxia, where the NOS system and aerobic performance are both limited. Our data, however, does not support this postulate. In our study, twelve male subjects completed four cycling trials consisting of a 15-minute warm-up, 15-minute steady-state cycling at 50% of maximum power output and a 10-km TT in a randomized protocol, with single-blind environmental condition (sea level or 2500 m) and double-blind supplementation (BR or PL). Our BR supplementation protocol had no effect on FENO or resting BP. Hypoxic exposure increased HR and VE and decreased steady-state SpO2 and TOI relative to normoxia, but there was no difference in any variables between BR and PL supplementation. Performance in 10-km TT was negatively affected by hypoxia, but there was no difference in 10-km TT performance measures between BR and PL trials. Overall, the lack of effect of BR in our study is likely attributable to relatively high subject fitness, the moderate severity of our hypoxic exposure, and our dosing protocol.  3.1 Conclusions regarding thesis hypotheses	 ? Hypothesis 1: I accept our hypothesis that BR would have no performance enhancing effect in normoxia. BR did not improve markers of exercise performance in normoxia.   Hypothesis 2: I reject our hypothesis that BR would improve these markers in hypoxia, relative to PL. BR did not improve markers of exercise performance in moderate hypoxia.   	 ? 43	 ?Hypothesis 3: I reject our hypothesis that BR would decrease resting blood pressure.  BR did not lower resting systolic or diastolic BP relative to PL following acute BR supplementation.   Hypothesis 4: I accept our hypothesis that FENO would increase after consumption of BR. FENO was significantly increased with BR compared to PL.   3.2 Strengths, limitations, future directions  This is the first study to compare the effect of BR supplementation on exercise tolerance and performance in trained athletes in normoxia and hypoxia in a double blind, randomized, crossover design. The BR product is commercially available and the acute supplementation protocol used is representative of realistic practices for athletes in training or at competition.  In designing the experiments, we chose to use a BR dose that falls within the recommended ?safe? guidelines for NO3- supplementation (Lundberg et al., 2011a).  Anecdotally, subjects could not differentiate between the BR and PL supplements, confirming the strength of the double blind.  During the course of this project, data was published indicating that there is a dose-response effect with BR (Wylie et al., 2013a) suggesting that the BR dose in this study was too small to have a significant effect on performance (although other studies have demonstrated performance improvement with a similar dose to ours, e.g., Muggeridge et al., (2014)). As Wylie et al. (2013a) reported no adverse events in their study with increasing NO3- dose; it may be necessary to revisit guidelines for what is a ?safe? acute dose. These authors also demonstrated that supplementing with BR containing 16.8 mmol NO3- resulted in no greater improvement to performance than BR containing 8.4 mmol NO3-. This conclusion is particularly relevant to athletes and warrants further investigation. Although we did not report any significant performance related results, other studies have shown BR to have a positive effect on performance. Variables affecting the efficacy of BR should be investigated. Future studies should also investigate the best BR dose relative to fitness. Realistically, each athlete will have a different optimal dose, possibly based on fitness and weight. BR supplementation protocols should be 	 ? 44	 ?developed on a case-by-case basis to balance maximized performance with potential risks. To this end, general BR supplementation guidelines and recommendations should also be developed as a starting point for athletes and coaches. The effect of BR during acute hypoxic exposures (from low to high altitude) and chronic altitude acclimatization should also be further investigated. Despite the lack of effect on performance in this study, the increase in FENO following BR supplementation indicates increased NO production. NO production has previously been suggested to be important for functional adaptation to hypoxia (Erzurum et al., 2007). Therefore, there may be beneficial effects at the molecular level that were not great enough to cause a statistically detectable change in exercise performance in this thesis study. Longitudinal studies assessing safety of long term NO3- supplementation are also important.  Part of the appeal of supplementing with BR products is that beets may be considered a ?functional food? (Reid 2013) that could enhance sport performance through consumption of a healthy diet. While this study focused specifically on whether the [NO3-] in our BR dose would improve performance, other components of BR juice have also been suggested as potential performance enhancers. For example, betaine is gaining interest in sport literature, and has been demonstrated to benefit repeated cycling sprint performance (Pryor et al., 2012). Beets also contain antioxidant compounds (Kanner et al., 2001), which may be helpful in recovery. It is possible that these components in beets work synergistically in the body, and are collectively involved in the overall performance enhancement that has been demonstrated with BR supplementation. As our BR and PL supplement were identical aside from NO3- concentration, this could have affected our results. 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We investigated the effect of BR on steady state economy and 10-km time trial (TT) performance in normoxia, and in hypoxia (simulated altitude of 2500 m). METHODS: Trained male cyclists (VO2max ? 60 ml?kg-1?min-1) completed four exercise trials. Two hours prior to exercise, subjects consumed 70 mL BR (~6 mmol nitrate) or 70 mL placebo (nitrate-depleted BR) in a randomized, double-blind manner. Subjects then completed a 15-min self-selected warm up followed by a 15-min steady-state bout at 50% max power output, and a 10-km time trial (TT) in either normoxia (~21% O2) or hypoxia (~16% O2). Environmental conditions were randomized and single-blind. Exercise economy (mL O2?min-1?W), oxygen saturation (SpO2), mean power output (MPO) and time to complete the 10-km TT were measured. RESUL TS : Economy at 50% power output was similar in hypoxic and normoxic conditions (mean [SD]: 14.5 [0.4] vs. 14.3 [1.2] mL O2?min-1?W; p > 0.05), but subjects had a significantly higher MPO in the normoxic TT (294 [44] W) relative to the hypoxic TT (246 [36] W; p < 0.05). BR did not affect economy (14.2 [1.4] vs. 14.3 [1.3] mL O2?min-1?W), SpO2 (98 [1.7] vs. 98 [1.4 %]), MPO (295 [42] vs. 293 [46] W) or time to complete the 10-km TT (941 [48] vs. 951 [58] s) relative to placebo in normoxia (p > 0.05 in all comparisons). Similarly, in hypoxia, BR did not affect economy (14.6 [1.3] vs. 14.3 [1.2] mL O2?min- 1?W), SpO2 (87 [1.9] vs. 87 [2.1] %), MPO (244 [35] vs. 247 [37] W) or time to complete the 10-km TT (1014 [61] vs. 1010 [55] s) relative to placebo (p > 0.05 in all comparisons). CONCLUSIONS: In a small sample of well-trained cyclists, BR did not improve exercise performance in normoxia or hypoxia; however, more subjects will need to be tested to confirm these conclusions if these data are to be applied to elite athletes.    	 ? 55	 ?    	 ? 56	 ?Appendix B  Table 8: Summary of studies using beetroot supplementation.  Author  Participants BR protocol Exercise Protocol Conclusions  Bailey et al., 2009   8 healthy, recreationally active young men  500 ml per day BR (11.2?0.6 mmol NO3-) x 6 days. Double blind, cross-over with cordial placebo  Moderate- and severe-intensity exercise tests on last three days of supplementation (including TTE), cycling.  BR reduced oxygen cost of exercise in both moderate and severe intensity, possibly related to improved mitochondrial efficiency. (Assessed muscle oxygen uptake kinetics)  Bailey et al., 2010  7 young men 500 ml per day of either BR (5.1 mmol NO3-) or PL for 6 consecutive days. Double blind, crossover Low- and high- intensity ?step? exercise tests over last 3 days of supplementation (knee extensor exercise) BR decreased: muscle ATP turnover rate, ADP accumulation, muscle Pi accumulation, PCr depletion, VO2 amplitude during low-intensity exercise, VO2 slow-component during high-intensity exercise-increased severe exercise limit of tolerance  Vanhatalo et al., 2010  8 healthy adults (5 males, 3 females) physically active  500 ml BR per day (5.2 mmol NO3-) or 500 ml low-calorie cordial placebo for 15 days 2 moderate-intensity step tests followed by ramp test repeated after 2.5 hrs following first ingestion, and after 5 and 15 days of BR and PL   Reduced BP, and O2 cost of submaximal exercise, can be maintained for 15 days if BR supplementation is continued. Kenjale et al., 2011  4 male and 4 female subjects; PAD patients Consumed either 500 mL BR or 500 mL orange juice (matched for calories, sugar, carbohydrates and protein) 120 min before second tests. Cross over Treadmill maximal cardiopulmonary test With BR subjects walked 18% longer before claudication pain onset, and had a 17% longer peak walking time, reduction in fractional O2 extraction at working tissue, decreased DBP at rest and SBP and HR during recovery from max exercise. No changes in endothelial function  Lansley et al., 2011a  9 club-level competitive male cyclists 500 ml BR (~6.2 mmol NO3-) or 500 ml PL (NO3- depleted BR) in a 4 km and 16.1 km TT BR increased mean PO for both TT distances, although VO2 values did not 	 ? 57	 ?cross over design 2.5 hours before TT  differ (higher PO for same VO2) Lansley et al., 2011b   9 healthy, physically active males 500 ml BR per day, containing ~6.2 mmol NO3- or 500 ml placebo for 6 days (double blind, crossover)  Treadmill VO2max Treadmill tests on days 4 (2x 6 min moderate-intensity running and one exhaustive bout of severe-intensity as a measure of exercise tolerance) & 5 (2x 6 min bouts moderate-intensity running and one 6 min bout of severe intensity running) for VO2 responses Day 6, incremental single-legged knee-extension exercise test  BR reduced SBP, and O2 cost of walking, mod-intensity and severe-int running. BR improved TTE during severe-intensity running. Mitochondrial oxidative capacity not different between BR and PL      Vanhatalo et al., 2011  7 males, 2 females, healthy and moderately trained in rec sport 750 mL BR (9.3 mmol NO3-) or 750 mL NO3- depleted BR (double blind, crossover) 24 hrs prior to hypoxia trials Knee-extension exercise to limit of tolerance?once in normoxia, twice in hypoxia (14.5% O2) Limit of tolerance reduced with hypoxia, but no difference between normoxic control and hypoxia+BR. BR reduced muscle metabolic perturbation [PCr, Pi, pH]. PCr recovery time was higher in hypoxia+PL vs hypoxia+BR and normoxia+control. Exercise tolerance and oxidative function restored with BR in hypoxia ? normoxia+control  Bond et al., 2012  14 well-trained male junior rowers  500 ml BR or PL for 6 days (7 day washout) 6 maximal 500 m ergometer repetitions  Improved max rowing-ergometer reps (particularly reps 4-6).  Cermak et al., 2012a   12 male cyclists 6 days of 140 ml per day (8 mmol NO3-) BR or NO3- depleted BR (double blind, crossover) 14 day washout 60 min submaximal cycling (2x 30 min at 45% and 65% max watts) followed by 10 km TT TT and PO improved with BR. Submaximal VO2 was lower with BR. Whole body fuel selection, plasma lactate, glucose and insulin did not differ between conditions  Cermak et al., 2012b   20 trained male cyclists 140 mL concentrated BR or 140 mL PL 2.5 hrs before TT. Randomized, double blind ~1 hour TT BR did not improve TT performance, or PO. Plasma NO2- higher following BR 	 ? 58	 ? Engan et al., 2012  12 healthy men and women, trained breath-hold divers 70 ml BR or nitrate depleted BR 2.5 hrs prior to testing Static apnea series, of two-min sub-max apneas separated by 3 min recovery, followed y 5 min recovery before final maximal effort apnea BR increased maximal apneic duration by 11%, significantly higher SaO2 for BR supplemented sub-max apneas, SaO2 lower with BR after max apneas (but longer duration), reduced MAP with BR   Hobbs et al., 2012   Study 1: 18 normotensive, healthy males Study 2: 14 healthy males  Study 1: randomized to receive either 500 g H20, 100 g BR with 400 g H20, 250 g BR with 250 g H20, or 500 g BR Study 2: randomized to receive either 200 g white bread, 200 g bread enriched with white beetroot (50% total weight), or 200 g bread enriched with red beetroot (50% total weight)   ABP measured every 15 minutes from 8 am-1 pm, every 30 minutes from 1-10 pm and every 60 minutes from 10 pm ? 8 am. Urine collected at baseline, 2 and 4 hrs post intervention and 24 hrs post  Nearly dose-dependent decrease in ABP with BR enriched products.  Masschelein et al., 2012  15 young, physically active males 6-day supplementation with 500 ml BR (0.07 mmol.kg-1 nitrate) or apple-blackcurrant juice in 5 equal doses throughout the day, and full dose 1-2 hrs pre-exercise tests on the final day (single-blind, cross over) 20 min submax constant load cycling at workload equal to 45% VO2peak, and an maximal exercise test to volitional exhaustion (one normoxic control test, and hypoxic+PL and hypoxic+BR tests equivalent to 5500 m) With BR, lower VO2 at rest and during 45% ex test, SpO2 higher. BR partly negated decreased TTE in hypoxia. BR improved muscle oxygenation but not cerebral oxygenation in severe hypoxia. BR improved exercise tolerance, but there were similar AMS symptoms with both BR and PL  Murphy et al., 2012  11 recreationally fit men and women   Randomized cross over, 200 g baked whole beetroot or cranberry relish 75 minutes pre-exercise trials  5-10 minutes self-paced treadmill warm up followed by 5 km running TT With BR: 3% faster TT, RPE lower over 1st 1.6 km  Wilkerson et al., 2012  8 well trained male cyclists 500 ml BR or 500 ml NO3- depleted BR 2.5 hrs before TT 50 mile TT PO did not differ between conditions, but VO2 tended to be lower in BR (p=0.06) resulting in a greater 	 ? 59	 ?PO/VO2 ratio. BR did not improve TT performance.  Bond et al., 2013a   12 healthy females Randomized cross over supplementation with either 500 mL BR or 500 mL orange juice Steady state submaximal cycling, with three successive stages of 5 min at 40%, 60% and 80% of VO2peak BR improved cerebral and systemic hemodynamics at rest and at 40% and 60% workloads  Bond et al., 2013b   12 healthy women  500 mL BR or orange juice placebo  Trials consisting of 40%, 60% and 80% of VO2peak submaximal exercise  BR decreased VO2, SBP and heart rate-systolic pressure product at rest, and at 40%, 60% and 80% of VO2peak     Christensen et al., 2013  10 elite trained male cyclists Cross over (with 14-day washout in between supplementation periods). 6-day supplement period of either 500 ml BR (~0.5% NO3-) or 500 ml isocaloric PL (apple & blackcurrant drink) Baseline VO2max. 4th day of supplementation: 2 x 6 min at 70 & iPPO separated by 60 min. Followed by RST of 6x20 s sprints separated by 100 s low-int cycling. 6th day suppl: 1 x 6 min at 70% iPPO, 5 min later an endurance tests consisting of 2-hr ?pre-load? (20 min at 50% iPPO, 10 min variable int avg of ~53% iPPO 4 times) followed by 400 kcal TT  No significant changes in VO2 kinetics, endurance capacity (no change in economy), and repeated sprint performance. Two responders benefitted from BR, with improved economy Fulford et al., 2013  8 active males Double blind, randomized cross over. Two-week washout. 15 days 500 mL per day BR or PL (NO3- depleted BR)   Short bouts of continuous exercise followed by a repetitive MVC protocol BR supplementation reduced PCr cost of contraction while maintaining muscle-force generating capacity Hobbs et al., 2013  23 healthy males 200 g bread containing 100 g BR or 200 g white bread, randomized, open-label, crossover Postprandial microvascular vasodilation, arterial stiffness and ambulatory BP measured over 6 hours BR bread increased endothelium-independent vasodilation and decreased DBP  Hoon et al., 2013a   10 highly-trained male rowers  Double blind crossover supplementation with PL (0 mmol NO3-), single BR (4.2 mmol), or double BR (8.4  2000 m rowing TT  Single BR demonstrated a negligible effect on TT performance. Double BR possibly beneficial to TT performance 	 ? 60	 ?mmol)  Kelly et al., 2013   6 male, 6 female healthy adults (60-70 yrs)  3 days with BR (2x70 ml) or PL (NO3- depleted BR) Double blind, randomized, cross over  BP, sub-maximal ramp incremental treadmill ex test, 2x 6 min walk tests (moderate intensity) followed by 10 min passive recovery. 3 cognitive tests  BR decreased resting BP (DBP/SBP/MAP), accelerated VO2 kinetics during treadmill walking (though there was no improvement during the 6 minute walk tests). Cognitive performance unaltered.    Lanceley et al., 2013   11 trained females runners  Double-blind, repeated measures crossover with 14 day washout. Supplementation with either BR (~8 mmol NO3-) or PL for 4 days and 2.5 hrs pre exercise  5 km TT  No main effect for BP,  although slightly lower SBP with BR. TT performance unchanged between trials, with a trend towards improvements with BR.   Lane et al., 2013    12 male and 12 female competitive cyclists  Double blind latin squares design. Supplementation with either caffeine (3 mg?kg-1), BR (8.4 mmol NO3-), BR + caffeine, or no supplementation (control)  Females: 29.35 km cycling TT  Males: 43.83 km cycling TT  Power output significantly increased with caffeine and caffeine + BR compared to control. BR not ergogenic alone or when combined with caffeine, compared to the caffeine trial. BR not ergogenic in this trial.  Liu et al., 2013    26 participants aged 38-69   Randomized cross over, high (220 mg NO3- from spinach) or low nitrate energy-matched meals  Blood pressure and arterial stiffness measurements pre- and up to 210 min post meal  Spinach resulted in higher large artery elasticity index, lower pulse pressure, and lower systolic BP  Muggeridge et al., 2013   8 male kayakers  Randomized, cross over design. 70 mL concentrated BR or tomato juice PL  3 x trials consisting of 15 min paddling at 60% max work rate, five x ten-sec sprints and a 1 k TT (2nd and 3rd trials with BR or PL)  No difference in peak power or TT performance. VO2 reduced. BR shows no effect with repeated supramaximal kayak sprints or 1 km TT performance.   Torre et al., 2013   7 healthy males  Randomized double-blind crossover supplementation with either 0.5 L/day spinach juice (5.5 mol/day NO3-)  Five ?all out? 6 second sprints on a cycle ergometer, separated by 24 seconds passive recovery  Absolute peak power not different between spinach and PL sprints 1 and 2. Peak power significantly greater during sprints 3, 4 and 5 with spinach 	 ? 61	 ?or PL      Wylie et al., 2013a    14 male, recreational team-sport players  Double-blind, randomized crossover 490 mL BR or NO3- depleted PL over 30 hrs preceding test Yo-Yo intermittent recovery level 1 test  Intermittent performance  improved with BR. Lower blood glucose concentration and reduced plasma potassium increase with BR  Wylie et al., 2013b  10 healthy males Randomized, cross over design. 70, 140 or 280 mL concentrated BR containing 4.2, 8.4 and 16.8 mmol NO3- respectively, or NO3- depleted PL  24 hr assessment of dose on plasma [NO3-] and [NO2-]. On separate days, moderate- and severe- intensity cycle exercise tests  Plasma [NO2-] increased in a dose-dependent matter. 140 mL and 280 mL BR reduced steady-state VO 2 by 1.7% and increased time to task failure significantly   Muggeridge et al., 2014   9 competitive male cyclists   70 mL concentrated BR or NO3- depleted PL  3 x trials consisting of 15 min steady state at 60% max work rate and a 16.1 km TT at simulated 2500 m  VO2 lower during steady state exercise, and TT performance significantly faster with BR       Note. BR: beetroot juice, PL: placebo, SBP: systolic blood pressure, DBP: diastolic blood pressure, ABP: ambulatory blood pressure, MAP: mean arterial pressure, I/R: ischemia reperfusion, VE: ventilation, RER: respiratory exchange ratio, PO: power output, RPE: rating of perceived exertion, PAD: peripheral arterial disease, iPPO: incremental test to peak power output, MVC: maximal voluntary contraction 	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?     	 ? 62	 ?Appendix C  Normoxic and hypoxic air: Subjects breathed air from a normobaric hypoxia chamber while seated on the cycle ergometer outside of the chamber, through a breathing tube attached to the facemask. On hypoxic test days, subjects breathed ~16% O2 from the chamber to simulate an altitude of ~2500 m. On normoxic control days, subjects breathed ~21% O2, which corresponded to sea level. In either condition, the sounds and set-up of the exposure were indistinguishable, helping to enhance the quality of the environmental single-blind. With subjects sitting outside of the altitude chamber, low CO2 concentrations could be maintained inside, as subjects exhaled to the metabolic cart outside the chamber. Analyzers were set up to continuously monitor FIO2 and FICO2 from the chamber, and these values were later used to correct metabolic cart calculations. To use the metabolic cart when subjects were breathing air from the hypoxia chamber, analyzers (17625 O2 Analyzer and 17630 CO2 Analyzer, Vacumed, Ventura, CA) were calibrated and set up to sample O2 and CO2 continuously from the chamber. Gas sampling data was collected with a Power Lab (PL3508 Powerlab, ADInstruments, Colorado Springs, CO), collected by a computer, and saved to a Lab Chart (LabChart V7, ADInstruments, Colorado Springs, CO) file. Temperature and humidity in the room were noted during calibration and were later used to calculate water vapour pressure to correct metabolic cart data. Because the metabolic cart calculations data during exercise assumed subjects breathed normoxic air, the data was later corrected. Fifteen-second mean FICO2 and FIO2 data from the hypoxia chamber was calculated to match with 15-second data intervals calculated by the metabolic cart. The metabolic cart calculates data based on dry air. Therefore, it was necessary to calculate (and remove) water vapour from our measured FIO2. Water vapour pressure during the test was calculated using a water vapour vs. temperature chart and the following calculations:  	 ? 63	 ?1. Water vapour pressure (PH2O in mmHg) was calculated by multiplying the pressure of aqueous vapour over water with humidity measured in the laboratory, divided by 100.  e.g., (adjusted temperature * humidity) /100 = PH2O 2. The fraction of the barometric pressure made up of water vapour pressure was calculated by dividing the result of the above calculation by barometric pressure in the laboratory, multiplied by 100. e.g., (PH2O / PB ) * 100 = X% 3. This fraction was then multiplied by the measured FIO2 in the chamber, divided by 100, to determine the fraction of water vapour in measured FIO2 e.g., X * FIO2  / 100 4. This value was then subtracted from the measured FIO2 to determine actual FIO2   Once the corrected FIO2 was calculated, the data provided by the cart needed to be adjusted. The following calculations were used to determine final metabolic data:  Fraction of inspired nitrogen (FIN2): = 1 - FIO2 / 100 - 0.0005  Fraction of expired nitrogen (FEN2): = 1 - FEO2 / 100 - FECO2 / 100  Ratio of inspired to expired nitrogen (FIN2/FEN2): = FIN2 / FEN2  Inspired volume (VI): = VE * FEN2 / FIN2  O2 (L?min-1): = (VI * FIO2 ? VE * FEO2) / 100  O2 STPD: = calculated O2 / BTPS to STPD conversion value (provided by cart)  O2 (ml?kg-1?min-1): = O2 STPD * 1000 / subject mass in kg  CO2: = (VE * FECO2 ? VI * FICO2) / 100  CO2 STPD: = CO2 / BTPS to STPD conversion value (provided by cart)  RER: = CO2 STPD/ O2 STPD  	 ?Note that in the above equations, fractions were entered as percentages, hence the frequent division by 100.      ?V?V ?V?V ?V?V?V ?V?V ?V	 ? 64	 ? Appendix D 	 ? Food	 ?and	 ?Training	 ?Log	 ?	 ?Today?s	 ?Date:	 ?(Day	 ?of	 ?testing)	 ?Food	 ?Consumption	 ?(please	 ?report	 ?the	 ?time	 ?of	 ?consumption	 ?as	 ?well):	 ?Morning	 ?(Breakfast/Snacks):	 ?	 ?	 ?	 ?	 ?Noon	 ?(Lunch/Snacks)	 ?	 ?	 ?	 ?	 ?	 ?Evening/Night	 ?(Dinner/Snacks)	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?Caffeine:	 ?YES/NO	 ?	 ?	 ?If	 ?yes,	 ?when:	 ?Physical	 ?Activity:	 ?(activity,	 ?duration	 ?and	 ?intensity)	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?Hours	 ?of	 ?Sleep:	 ?	 ?	 ?	 ?	 ?	 ? 65	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?One	 ?day	 ?prior	 ?to	 ?testing:	 ?Food	 ?Consumption	 ?(please	 ?report	 ?the	 ?time	 ?of	 ?consumption	 ?as	 ?well):	 ?Morning	 ?(Breakfast/Snacks):	 ?	 ?	 ?	 ?	 ?	 ?	 ?Noon	 ?(Lunch/Snacks)	 ?	 ?	 ?	 ? 	 ?	 ?	 ?Evening/Night	 ?(Dinner/Snacks)	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ? Physical	 ?Activity:	 ?(type,	 ?duration	 ?and	 ?intensity)	 ?	 ?	 ?	 ?	 ? 	 ?	 ? 66	 ?	 ?	 ?	 ?	 ?No changes permitted. You are encouraged to photocopy the PAR-Q but only if you use the entire form.1. Has your doctor ever said that you have a heart condition and that you should only do physical activity recommended by a doctor?2. Do you feel pain in your chest when you do physical activity?3. In the past month, have you had chest pain when you were not doing physical activity?4. Do you lose your balance because of dizziness or do you ever lose consciousness?5. Do you have a bone or joint problem (for example, back, knee or hip) that could be made worse by a change in your physical activity?6. Is your doctor currently prescribing drugs (for example, water pills) for your blood pressure or heart con-dition? 7. Do you know of any other reason why you should not do physical activity?PLEASE NOTE:  If  your health changes so that you then answer YES to any of  the above questions, tell your fitness or health professional.   Ask whether you should change your physical activity plan.Regular physical activity is fun and healthy, and increasingly more people are starting to become more active every day.  Being more active is very safe for most people. However, some people should check with their doctor before they start becoming much more physically active.If  you are planning to become much more physically active than you are now, start by answering the seven questions in the box below.  If  you are between the ages of  15 and 69, the PAR-Q will tell you if  you should check with your doctor before you start.  If  you are over 69 years of  age, and you are not used to being very active, check with your doctor.Common sense is your best guide when you answer these questions.  Please read the questions carefully and answer each one honestly:  check YES or NO.Talk with your doctor by phone or in person BEFORE you start becoming much more physically active or BEFORE you have a fitness appraisal.  Tell your doctor about the PAR-Q and which questions you answered YES.? <RXPD\EHDEOHWRGRDQ\DFWLYLW\\RXZDQW?DVORQJDV\RXVWDUWVORZO\DQGEXLOGXSJUDGXDOO\2U\RXPD\QHHGWRUHVWULFW\RXUDFWLYLWLHVWRthose which are safe for you. Talk with your doctor about the kinds of  activities you wish to participate in and follow his/her advice.? )LQGRXWZKLFKFRPPXQLW\SURJUDPVDUHVDIHDQGKHOSIXOIRU\RXPAR-Q & YOU?Physical Activity ReadinessQuestionnaire - PAR-Q  (revised 2002)DELAY BECOMING MUCH MORE ACTIVE:? LI \RXDUHQRWIHHOLQJZHOOEHFDXVHRI DWHPSRUDU\LOOQHVVVXFKDVa cold or a fever ? wait until you feel better; or? LI \RXDUHRUPD\EHSUHJQDQW?WDONWR\RXUGRFWRUEHIRUH\RXstart becoming more active.If  you  answered If  you answered NO honestly to all PAR-Q questions, you can be reasonably sure that you can:? VWDUWEHFRPLQJPXFKPRUHSK\VLFDOO\DFWLYH?EHJLQVORZO\DQGEXLOGXSJUDGXDOO\7KLVLVWKHsafest and easiest way to go.? WDNHSDUWLQDILWQHVVDSSUDLVDO?WKLVLVDQH[FHOOHQWZD\WRGHWHUPLQH\RXUEDVLFILWQHVVVRthat you can plan the best way for you to live actively. It is also highly recommended that you have your blood pressure evaluated.  If  your reading is over 144/94, talk with your doctor before you start becoming much more physically active.NOTE:  If  the PAR-Q is being given to a person before he or she participates in a physical activity program or a fitness appraisal, this section may be used for legal or administrative purposes."I have read, understood and completed this questionnaire.  Any questions I had were answered to my full satisfaction."NAME ________________________________________________________________________  SIGNATURE _______________________________________________________________________________  DATE ______________________________________________________SIGNATURE OF PARENT  _______________________________________________________________________  WITNESS ___________________________________________________or GUARDIAN (for participants under the age of  majority)Informed Use of  the PAR-Q:  The Canadian Society for Exercise Physiology, Health Canada, and their agents assume no liability for persons who undertake physical activity, and if  in doubt after completing this questionnaire, consult your doctor prior to physical activity.(A Questionnaire for People Aged 15 to 69) YES NOYES to one or more questionsNO to all questionsNote:  This physical activity clearance is valid for a maximum of 12 months from the date it is completed and  becomes invalid if your condition changes so that you would answer YES to any of the seven questions.? Canadian Society for Exercise Physiology  www.csep.ca/forms	 ? 67	 ?Physical	 ?Activity	 ?History	 ?1.	 ?Type	 ?of	 ?physical	 ?activity	 ?(please	 ?list	 ?all	 ?sports/exercises	 ?you	 ?participate	 ?on	 ?a	 ?regular	 ?basis)	 ?Type	 ?of	 ?sport	 ? Volume	 ?(a).	 ?how	 ?many	 ?minutes/session	 ?and	 ?(b)	 ?how	 ?many	 ?times/week?)	 ?	 ? a)	 ?b).	 ?	 ? a)	 ?b).	 ?	 ? a).	 ?b).	 ?	 ? a).	 ?b).	 ?	 ? a).	 ?b).	 ?	 ?2.	 ?How	 ?long	 ?have	 ?you	 ?been	 ?a	 ?competitive	 ?cyclist/triathlete?	 ?_____________________________	 ?3.	 ?Volume	 ?of	 ?cycling	 ?per	 ?week:	 ?	 ?__________hours/week	 ?	 ?	 ? 	 ? 	 ? 	 ?	 ?	 ?	 ?__________km/week	 ?(solely	 ?for	 ?cycling)	 ?4.	 ?Are	 ?you	 ?in-??season	 ?or	 ?off-??season	 ?currently?	 ?	 ?IN-??SEASON/	 ?OFF-??SEASON	 ?5.	 ?Highest	 ?level	 ?of	 ?competition:____________________________________________________	 ?6.	 ?	 ?VO2max	 ?(if	 ?known):	 ?__________;	 ?Date:_________;	 ?Modality	 ?(bike,	 ?treadmill)___________	 ?7.	 ?Last	 ?cycling	 ?race:	 ?_____________;	 ?Distance:____________;	 ?Date:______________________	 ?8.	 ?Cycling	 ?category	 ?(if	 ?applicable,	 ?please	 ?list	 ?how	 ?many	 ?years	 ?you?ve	 ?competed	 ?in	 ?each	 ?category)	 ?______________________________________________________________________________	 ?	 ?	 ? 	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ? 68	 ?Appendix E   Fig 8: Heart rate (HR) during steady state cycling at 50% maximum power output in hypoxia and normoxia when supplemented with placebo (PL) or beetroot juice (BR). Normoxic and hypoxic trials were significantly different (P < 0.001). Error bars represent standard deviation.       Fig 9: Pulse oxygen saturation (SpO2) during steady state cycling at 50% maximum power output in hypoxia and normoxia when supplemented with placebo (PL) or beetroot juice (BR). Normoxic and hypoxic steady state were significantly different (P < 0.001). Error bars represent standard deviation.      HR  (bpm)Normoxia Hypoxia050100150200PLBRP<0.001SPO2 (%)Normoxia Hypoxia020406080100PLBRP<0.001	 ? 69	 ? Fig 10: Pulmonary ventilation (VE) during steady state cycling at 50% of maximum power output in hypoxia and normoxia when supplemented with placebo (PL) or beetroot juice (BR). Hypoxic and normoxic steady state were significantly different (P < 0.001). Error bars represent standard deviation.      Fig 11: Individual subject economy (mL O2?min-1?W-1) data during steady state cycling at 50% of maximum power output in normoxia when supplemented with placebo (PL) or beetroot juice (BR).            Ventilation (L? min-1)Normoxia Hypoxia050100150PLBRP<0.001Economy (mL O2? min-1?  W-1)PL BR1012141618	 ? 70	 ? Fig 12: Individual subject economy (mL O2?min-1?W-1) data during steady state cycling at 50% of maximum power output in hypoxia when supplemented with placebo (PL) or beetroot juice (BR).                              Economy (mL O2? min-1?  W-1)PL BR1012141618	 ? 71	 ?Appendix F   Fig 13: Time (s) to complete a 10-km time trial in normoxia and hypoxia when supplemented with either placebo (PL) or beetroot juice (BR). Hypoxic and normoxic trials were significantly different (P < 0.001). Error bars represent standard deviation.     Fig 14: Individual subject data for the 10-km time trial (s) in normoxia when supplemented with either placebo (PL) or beetroot juice (BR).   10-km time (s)Normoxia Hypoxia050010001500PLBRP<0.00110-km time (s)PL BR800900100011001200	 ? 72	 ? Fig 15: Individual subject data for the 10-km time trial (s) in hypoxia when supplemented with either placebo (PL) or beetroot juice (BR).    Fig 16: Individual subject data for mean power output (W?kg-1) during the 10-km time trial in normoxia when supplemented with either placebo (PL) or beetroot juice (BR).  10-km time (s)PL BR800900100011001200Mean power (W? kg-1 )PL BR2.53.03.54.04.55.0	 ? 73	 ? Fig 17: Individual subject data for mean power output (W?kg-1) during the 10-km time trial in hypoxia when supplemented with either placebo (PL) or beetroot (BR).                                Mean power (W? kg-1 )PL BR2.53.03.54.0

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