Content uploaded by Charles R Pedlar
Author content
All content in this area was uploaded by Charles R Pedlar on Sep 19, 2021
Content may be subject to copyright.
. . . Published ahead of Print
A Single Dose of Beetroot Juice Enhances Cycling Performance in Simulated
Altitude
David J. Muggeridge1, 2, Christopher C.F. Howe2, Owen Spendiff2, Charles Pedlar3,
Philip E. James4, and Chris Easton1, 2
1Institute for Clinical Exercise and Health Science, University of the West of Scotland,
Hamilton, United Kingdom; 2School of Life Sciences, Kingston University, Kingston upon
Thames, United Kingdom; 3School of Sport, Health and Applied Science, St Mary’s University
College, Twickenham, United Kingdom; 4Wales Heart Research Institute, Cardiff University
Medical School, Cardiff, United Kingdom
Accepted for Publication: 24 June 2013
Medicine & Science in Sports & Exercise® Published ahead of Print contains articles in unedited
manuscript form that have been peer reviewed and accepted for publication. This manuscript will undergo
copyediting, page composition, and review of the resulting proof before it is published in its final form.
Please note that during the production process errors may be discovered that could affect the content.
Copyright © 2013 American College of Sports Medicine
ACCEPTED
A Single Dose of Beetroot Juice Enhances Cycling Performance in Simulated
Altitude
David J. Muggeridge1, 2, Christopher C.F. Howe2, Owen Spendiff2, Charles Pedlar3, Philip E.
James4, and Chris Easton1, 2
1Institute for Clinical Exercise and Health Science, University of the West of Scotland,
Hamilton, United Kingdom; 2School of Life Sciences, Kingston University, Kingston upon
Thames, United Kingdom; 3School of Sport, Health and Applied Science, St Mary’s University
College, Twickenham, United Kingdom; 4Wales Heart Research Institute, Cardiff University
Medical School, Cardiff, United Kingdom
Running Title: Nitrate and cycling performance at altitude
Address correspondence to: Dr Chris Easton BSc, PhD, FHEA
University of the West of Scotland
Almada Street
Hamilton, ML3 0JB, UK
E-mail: chris.easton@uws.ac.uk
Tel: (+44) 1698 283100 ext 8282
Fax: N/A
Conflicts of Interest and Source of Funding: None declared
Medicine & Science in Sports & Exercise, Publish Ahead of Print
DOI: 10.1249/MSS.0b013e3182a1dc51
ACCEPTED
Copyright © 2013 by the American College of Sports Medicine. Unauthorized reproduction of this article is prohibited.
ABSTRACT
Increasing nitric oxide bioavailability via supplementation with nitrate-rich beetroot juice (BR)
has been shown to attenuate the negative impact of hypoxia on peripheral oxygen saturation and
exercise tolerance.
Purpose: We investigated the effects of a single dose of concentrated BR on the physiological
responses to submaximal exercise and time trial (TT) performance in trained cyclists exposed to
moderate simulated altitude (~2500m).
Methods: Nine competitive amateur male cyclists (age 28 ± 8 yr, VO2peak at altitude 51.9 ± 5.8
mL·kg-1·min-1) completed four exercise trials consisting of an initial graded test to exhaustion and
three performance trials on a cycle ergometer. The performance trials comprised 15 min
submaximal steady-state exercise at 60% maximum work rate and a 16.1 km TT. The second and
third trials were preceded by ingestion of either 70 ml BR or nitrate-depleted BR (PLA) 3 h prior
to exercise.
Results: Plasma nitrate (PLA: 39.1 ± 3.5 μM, BR: 150.5 ± 9.3 μM) and nitrite (PLA: 289.8 ±
27.9 nM, BR: 678.1 ± 103.5 nM) measured immediately prior to exercise, were higher following
ingestion of BR compared to PLA (P < 0.001, P = 0.004). VO2 during steady-state exercise was
lower in the BR trial (2542 ± 114 ml·min-1) than in the PLA trial (2727 ± 85 ml·min-1, P = 0.049).
TT performance was significantly faster following BR (1664 ± 14 s) than PLA (1702 ± 15 s, P =
0.021).
Conclusion: A single dose of BR lowered VO2 during submaximal exercise and enhanced TT
performance of trained cyclists in normobaric hypoxia. Consequently, ingestion of BR may be a
practical and effective ergogenic aid for endurance exercise at altitude.
Key Words: Nitrate; nitrite; supplementation; hypoxia; exercise
ACCEPTED
Copyright © 2013 by the American College of Sports Medicine. Unauthorized reproduction of this article is prohibited.
INTRODUCTION
Dietary nitrate supplementation results in an increase in plasma nitrate and nitrite via a nitric
oxide synthase (NOS) independent pathway and has been shown to reduce resting blood pressure
(22,39), attenuate the oxygen demand of submaximal exercise (1,2,23,25) and improve cycling,
running and rowing performance (6,8,24). Following ingestion of sodium nitrate or nitrate-rich
beetroot juice (BR), nitrate is reduced to nitrite, initially by bacteria in the gut and subsequently
by commensal bacteria in the oral cavity after re-entering the mouth via the entero-salivary
system. Following this process the nitrite is further reduced to nitric oxide (NO) in the acidic
conditions of the stomach. However, some nitrite survives this process and is absorbed by the
intestines into the systemic circulation. This circulating nitrite is subsequently reduced to
bioactive NO when hypoxic (7) and acidic (29) conditions are prevalent within the cell. The
consequences of an increased NO concentration may include an increase in muscle blood flow
and regulation of muscular contractions, glucose uptake and cellular respiration (36).
NO also plays an essential role in the physiological response to acute and chronic altitude
exposure. For example, when native lowlanders ascend to altitude they typically experience a
reduction in exhaled NO (suggesting a reduced NO production), the extent of which may be
associated with the prevalence of altitude sickness (10,11). The purported mechanism for this
reduced NO production may be an increase in oxidative stress as a consequence of hypoxia
and/or an inactivation of endogenous NOS (eNOS) that catalyses NO from circulating L-arginine
(27). The reduction in the partial pressure of arterial oxygen (PO2) and consequent tissue hypoxia
resulting from altitude exposure also have a profound ergolytic effect on endurance exercise
tolerance and physical performance that is due in part to a disturbance in muscle metabolism
(29). However, individuals who have adapted to living at high altitude have a higher
ACCEPTED
Copyright © 2013 by the American College of Sports Medicine. Unauthorized reproduction of this article is prohibited.
concentration of NO products, including plasma nitrate and nitrite, than lowland based controls
(14). These individuals appear to maintain basal and maximal oxygen consumption rates that are
consistent with sea level residents due to a greater blood flow associated with the increased
production of NO (3,19). It is possible that this adaptation occurs due to an increased availability
of intracellular L-arginine causing greater endogenous NO synthesis (14) although the precise
mechanism is presently unclear. Nevertheless, these adaptations are of limited benefit to
individuals who are acutely exposed to hypoxic environments during athletic or sporting
competitions. Given that endurance events such as the mountain stages in cycling tours are
regularly held at altitude, it is logical to assume that any alternative method to increase the
concentration of NO would be of benefit to these athletes.
Intriguingly, increased NO production as a direct consequence of dietary nitrate
supplementation, may offset the reduction in NO during hypoxia and minimize the negative
consequences on exercise performance. For example, Vanhatalo et al. (38) demonstrated that
ingestion of BR reduced muscle metabolic perturbation and enhanced exercise tolerance during
leg extension exercise when performed under hypoxic conditions (14.5% O2). Masschelein et al.
(28) also investigated exercise tolerance in cyclists during exposure to severe normobaric
hypoxia (11% O2) following a chronic (6-day) supplementation period of dietary nitrate and
found an improvement in peripheral oxygen saturation (SpO2) that was associated with extended
time to exhaustion in a maximal incremental exercise test. Despite this, no study has yet
determined the effects of an acute dose of BR on the oxygen cost of submaximal exercise and
performance at moderate altitude. Therefore, the aim of this study was to investigate the effects
of a single dose of BR on the oxygen cost, peripheral oxygen saturation and time trial (TT)
ACCEPTED
Copyright © 2013 by the American College of Sports Medicine. Unauthorized reproduction of this article is prohibited.
performance of trained cyclists exposed to acute normobaric hypoxia in order to simulate
moderate altitude.
METHODS
Participants
Nine male trained cyclists (age 28 ± 8 years, stature 182 ± 8 cm, body mass 77.7 ± 14.1 kg, and
VO2peak (determined at a simulated altitude of ~2500 m) 51.9 ± 5.8 mL·kg-1·min-1) volunteered
and provided written informed consent to participate in the study that was approved by the
Faculty of Science, Engineering and Computing Ethics Committee at Kingston University.
Participants were recruited from local cycling and triathlon clubs and were classified as trained
or well-trained based on cycling training and race status criteria proposed by Jeukendrup et al.
(21). The participants were all non-elite cyclists but regularly completed specific cycling training
(at least 3 sessions per week) and took part in competitive races including road races, TTs and
triathlons. All procedures were conducted in accordance with the Declaration of Helsinki.
Experimental Design
Each participant visited the laboratory on four separate occasions. On their first visit they
completed a maximal incremental test to exhaustion for determination of VO2peak and maximum
work rate (WRmax) in a normobaric hypoxic chamber set to a simulated altitude of ~2500 m (15%
O2) (Everest Summit II, Hypoxico, USA). The hypoxic chamber was fitted with an alarmed
sensor set so that the ambient fraction of inspired oxygen (FiO2) did not fall below 14.9% or rise
above 15.1% throughout the duration of any trial. Participants were not exposed to the hypoxic
environment of the chamber at any stage until approximately 5 min prior to the start of each
ACCEPTED
Copyright © 2013 by the American College of Sports Medicine. Unauthorized reproduction of this article is prohibited.
exercise trial. Each participant then completed three performance trials in the same
environmental conditions at the same time of day, at least five days apart. The first performance
trial consisted of a baseline measurement of performance in hypoxia with no supplementation
and the remaining two trials were preceded by ingestion of either BR (70 ml of concentrated
nitrate-rich BR [~5 mmol nitrate], Beet IT, James White Drinks Ltd, Ipswich) or a nitrate-
depleted placebo of BR (PLA) (Beet IT, James White Drinks Ltd, Ipswich [~0.01 mmol nitrate])
3 h prior to the start of exercise in a double-blind randomized cross-over design.
Pharmacokinetic data suggest that plasma nitrite will peak 2.5 ‒ 3 h after ingestion of a single
dose of BR (39) and this method of nitrate delivery has previously been demonstrated to enhance
exercise performance (24). Both supplements were identical in taste and packaging and therefore
neither participants nor lead investigators were able to identify which supplement had been
ingested. Participants were asked to follow their normal diet and activity patterns, although they
were requested not to exercise or consume alcohol for 24 h prior to each test, consume caffeine
for 6 h or to consume anything other than water for 3 h prior to testing.
Experimental Procedures
Maximal Exercise Test
Following standard anthropometric measurements, VO2peak and WRmax were measured using a
continuous graded exercise test on an electronically braked cycle ergometer (Velotron cycles,
Racermate Inc., USA). Participants performed an initial warm up consisting of cycling at 50 W
for 5 min followed by 5 min of static stretching. Subsequently, the exercise test commenced at an
initial workload of 50 W after which the work rate increased by 30 W every minute until
volitional exhaustion. Throughout the test SpO2 was continuously measured via a pulse oximeter
ACCEPTED
Copyright © 2013 by the American College of Sports Medicine. Unauthorized reproduction of this article is prohibited.
(BCI Autocorr, Smiths Medical, USA), WR and cadence were continuously monitored using
device software (CS 1.5 software), heart rate was measured by telemetry (Polar Electro, Oy,
Finland) and respiratory variables were measured via indirect calorimetry. Testing for the initial
batch of participants was completed using the Oxycon Pro metabolic cart (Jaeger, Hoechberg,
Germany) (n=7), however due to a malfunction it was replaced with the K4b2 portable metabolic
analyzer (Cosmed, Rome, Italy) (n=2). The same analyzer was used for the different
performance trials of each individual participant. We have previously shown that there is no
difference in the measurement accuracy of respiratory variables between the two analyzers in our
lab (12). Each metabolic analyzer was calibrated immediately prior to the test. The K4b2 was
calibrated inside the hypoxic chamber after the FiO2 setting had been altered to 15% using the
device software. The Oxycon Pro had the high/low FiO2 setting enabled on the device software
and was calibrated outside of the environmental chamber in accordance with the manufacturer’s
guidelines.
Performance Trials
Approximately one week after the maximal exercise test, each participant completed the first of
three separate cycling specific hypoxic performance trials. These consisted of 15 min continuous
steady-state cycling at 60% of WRmax and, following a 5 min passive rest period, a 16.1 km TT
(Velotron 3D software, version 1). During the TT each participant was instructed to cycle at a
freely chosen velocity and encouraged to complete the 16.1 km in the shortest time possible.
Participants received verbal feedback on the distance they had completed at 1 km intervals and
every 100 m for the last km. Heart rate, SpO2 and respiratory variables were continuously
monitored throughout each trial as previously described.
ACCEPTED
Copyright © 2013 by the American College of Sports Medicine. Unauthorized reproduction of this article is prohibited.
Blood collection and Analysis
Prior to the start of each performance trial participants were required to remain in the supine
position for 10 min after which blood pressure of the brachial artery was measured manually
using a stethoscope and sphygmomanometer (Accoson, London, UK) and 4 ml of venous blood
was collected from the cephalic vein. The blood was collected in a tube containing EDTA and
immediately centrifuged at 4000 rpm at 4oC for 10 min (1). The plasma was then separated into
two cryovials and immediately frozen in liquid nitrogen before being stored at -80oC for a
maximum of 4 months for later analysis of nitrate and nitrite via ozone-based
chemiluminescence (32). The procedures for the determination of nitrate and nitrite have been
previously described by Peacock et al. (31). Briefly, after samples were thawed in a water bath at
37°C for 3 min, nitrate concentration was determined using the reductant vanadium chloride in
hydrochloric acid at 80°C. Nitrite was determined in a separate assay via use of the reductant
potassium iodide in acetic acid at 50°C.
Data Analysis
Data are reported as mean ± SEM. Differences in blood pressure, plasma nitrate and nitrite, and
TT completion time between PLA and BR conditions were assessed using a paired samples t-
test. The remaining data were analyzed using a two-factor within-subjects repeated measures
ANOVA to examine the effects of supplement (BR or PLA), time and the interaction between
the two. Post-hoc analysis was completed using Bonferroni multiple comparisons. The
relationships between TT performance and plasma nitrate and nitrite concentrations were
assessed using Pearson’s correlation coefficient. The null hypothesis was rejected when P < 0.05.
Effect size (Cohens d) and 95% confidence intervals (95% CI) are included together with P
ACCEPTED
Copyright © 2013 by the American College of Sports Medicine. Unauthorized reproduction of this article is prohibited.
values, where appropriate. All statistical procedures were completed using SPSS for Mac version
19.0.
RESULTS
Plasma nitrate and nitrite and blood pressure
Plasma nitrate concentration in the baseline trial (42.8 ± 3.9 μM) did not change following PLA
(39.1 ± 3.5 μM, P = 0.482), but increased significantly following BR ingestion (150.5 ± 9.3 μM,
P < 0.001, 95% CI 89.3 – 133.1 μM) (Fig. 1). Plasma nitrite also increased significantly after BR
ingestion compared to baseline (P = 0.004, 95% CI 165.6 – 611.1 nM) but was not affected by
PLA (P = 0.160) (Baseline: 408.5 ± 59.0 nM, PLA: 289.8 ± 27.9, BR: 678.1 ± 103.5 nM) (Fig.
2). Systolic blood pressure was reduced following BR (PLA: 123 ± 7 mmHg, BR: 120 ± 5
mmHg, P = 0.041, 95% CI 0.15 – 5.85 mmHg). Diastolic blood pressure (DBP) and mean
arterial pressure (MAP) also tended to be lower although there was no statistical difference
between trials (DBP PLA: 76 ± 5 mmHg, BR: 74 ± 5 mmHg, P = 0.164; MAP PLA: 91 ± 5
mmHg, BR: 90 ± 3 mmHg, P = 0.089).
15-minute submaximal exercise
During the 15 min of submaximal steady-state exercise VO2 was significantly lower in the BR
trial compared to PLA (P = 0.049, 95% CI 1.3 – 369.5 ml·min-1, Fig. 3). Post-hoc analysis
revealed that VO2 was significantly reduced at the 12 min interval (P = 0.033, CI 22.3 – 393.9
ml·min-1) and 15 min interval (P = 0.049, CI 0.5 – 316.3 ml·min-1). There was no difference in
SpO2 between trials (P = 0.137, Fig. 4).
ACCEPTED
Copyright © 2013 by the American College of Sports Medicine. Unauthorized reproduction of this article is prohibited.
16.1 km Time Trial
BR improved performance by 2.9% compared to baseline (Baseline: 1716 ± 17 s; BR: 1664 ± 14
s, P = 0.006, CI 15.3 – 66 s) with a medium effect size ( d = 0.67) and performance was
significantly improved compared to PLA (PLA: 1702 ± 15 s, P = 0.021, Fig. 5). Performance
was not different between baseline and PL trials (P = 0.165). Eight of the nine participants were
quicker during the BR trial than the PLA trial (Fig. 5). Mean PO during the TT was not different
between baseline and PLA trials (Baseline: 212 ± 6 W; PLA: 216 ±6 W, P = 0.153) however
increased significantly following BR supplementation (BR: 224 ± 6 W, P = 0.021, 95% CI 4 –
19 W). There was no correlation between baseline plasma nitrite concentration and TT
performance in either the PLA (R = 0.030, P = 0.940) or BR trials (R = 0.523, P = 0.149). There
were also no correlation between the change in plasma nitrite, nitrate and submaximal VO 2 and
the change in TT performance between PLA and BR conditions (nitrite: R = –0.420, P = 0.227;
nitrate: R = 0.210, P = 0.587; VO2: R = –0.109, P = 0.781).
DISCUSSION
The deleterious impact of a hypoxic environment on endurance exercise performance is a major
issue for many athletes. Competitions are regularly held in moderate and high altitude
environments such as the mountain stages in the Tour de France (up to ~2800 m) and the Everest
Marathon (up to ~5200 m). The present study investigated the effects of acute BR ingestion on
cycling performance in normobaric hypoxic conditions. The principal finding was that a single
dose of BR reduced the oxygen cost of steady-state exercise and enhanced 16.1 km TT
performance at simulated altitude. Therefore, ingestion of BR prior to competition may provide a
ACCEPTED
Copyright © 2013 by the American College of Sports Medicine. Unauthorized reproduction of this article is prohibited.
simple but effective strategy to minimize the ergolytic effects of altitude exposure on endurance
exercise performance.
Other recent studies have also investigated the effects of dietary nitrate supplementation on the
response to exercise in hypoxia. Simulated altitude has been shown to have a profound ergolytic
effect on exercise tolerance, demonstrated by a 36% reduction in cycling time to exhaustion in
hypoxic conditions compared to normoxic conditions (28). The authors suggest that this may be
due to a reduction in arterial PO2 resulting in impaired O2 diffusion to the muscle. However, the
same authors demonstrated that supplementation with dietary nitrate, although not affecting
cerebral oxygenation and symptoms of acute mountain sickness, reduced VO2 and enhanced both
muscle oxygenation status and exercise time to exhaustion during cycling exercise at a simulated
altitude of 5,000m. Furthermore, Vanhatalo et al. (38) reported that BR reduced muscle
metabolic perturbation, demonstrated by a reduction in the rate of phosphocreatine (PCr)
degradation and Pi accumulation, and restored both exercise tolerance and oxidative function
during knee-extension exercise at a simulated altitude of 3,000m compared to that observed in
normoxia. The novel data obtained in the present study suggest that a single bolus of nitrate rich
BR with ensuing reduction in VO2 during submaximal exercise also translates to an enhancement
in actual exercise performance. This is despite no correlation between the reduction in VO2 and
the improvement in TT performance between PLA and BR conditions. The enhancement of
exercise performance following acute BR ingestion is consistent with some (6,8,24) but not all
(5,9,30,31,40) previous research in this area. Nevertheless, the mechanism(s) underpinning this
ergogenic effect continue(s) to be debated.
One possible explanation for the ergogenic effects of nitrate supplementation in this and other
studies (6,8,24) may be an augmented muscle blood flow during exercise. NO is a potent
ACCEPTED
Copyright © 2013 by the American College of Sports Medicine. Unauthorized reproduction of this article is prohibited.
vasodilator and nitrate supplementation has been shown to increase estimated local blood volume
at the muscle during unloaded cycling and the initial 120 s of moderate intensity exercise (1).
The authors attributed these effects to an enhanced muscle vasodilatation resulting from
increased NO production from nitrite. Ferguson et al. (16) demonstrated that blood flow and
vascular conductance in the exercising muscle of rats was higher following 5 days of BR
supplementation. Intriguingly, the increased blood flow and vascular conductance were observed
primarily in fast-twitch type II muscle fibres, suggesting the effects of dietary nitrate
supplementation may be fibre type selective. In contrast, Masschelein et al. (28) reported no
difference in regional blood volume in humans during submaximal and maximal exercise at
simulated altitude between BR and control conditions. Blood volume was estimated by
measuring the change in the fraction of total hemoglobin using near-infrared spectroscopy; a
measurement that correlates well with changes in tissue blood flow (37). Although blood flow
was not measured in the present study, these data suggest that the enhanced exercise
performance following nitrate supplementation cannot exclusively be explained by a stimulation
of local vasodilation and oxygen delivery to the muscle.
Instead, there is more compelling evidence to suggest that dietary nitrate supplementation may
improve the efficiency of mitochondrial respiration (26) and/or reduce the ATP cost of muscle
force production (2). When ATP is resynthesized via oxidative phosphorylation, there is a
leakage of protons back across the inner mitochondrial membrane into the mitochondrial matrix
from the intermembrane space. This protein leakage results in a substantial utilization of oxygen
(~25%) that does not contribute to ATP synthesis and accounts for 15% of active and 20% of
resting VO2 (33). Remarkably, Larsen et al. (26) reported that supplementation with dietary
nitrate can reduce proton leakage and the resulting improvement in mitochondrial efficiency may
ACCEPTED
Copyright © 2013 by the American College of Sports Medicine. Unauthorized reproduction of this article is prohibited.
explain, at least in part, the reduced oxygen cost of exercise. Consistent with this hypothesis was
their finding that the in vitro mitochondrial phosphate to oxygen ratio was reduced following
dietary nitrate. This was correlated with the reduction in the in vivo power output to VO2 ratio
during exercise suggesting enhanced efficiency of ATP synthesis. Alternatively, Bailey et al. (2)
suggest that the reduced oxygen cost of exercise following nitrate may be directly related to a
reduced ATP cost of cross-bridge cycling and/or calcium handling. Likewise, this hypothesis is
underpinned by a sound physiological mechanism as NO has been shown to modulate Ca2+
activation and the actin-myosin interaction during submaximal activation of skeletal muscle (20).
Indeed, supplementation with dietary nitrate has been shown to increase the myoplasmic free
[Ca2+] during tetanic stimulation of isolated mouse fast-twitch muscles leading to an enhanced
contractile force (18). The purported mechanism(s) accounting for the contribution of NO to
energy metabolism also indicate that supplementation with dietary nitrate may be particularly
pertinent when muscle oxygenation is compromised during hypoxia.
Data from the present study showed that ingestion of BR also resulted in a small increase in
SpO2 compared to the PLA condition although differences did not reach statistical significance
(Fig. 4). Masschelein et al. (28) have recently reported that dietary nitrate supplementation
resulted in a significant increase in SpO2 during exercise in severe hypoxia (11% ambient O2). It
is important to note that the SpO2 during submaximal exercise in the study of Masschelein et al.
(28) (~70%) was substantially lower than in the present study (~84%), presumably due to the
differences in FiO2 (11% vs. 15%, respectively). Additionally, these authors reported that the
muscle tissue oxygenation index, which assesses the fraction of oxygen saturated tissue
hemoglobin and myoglobin, was significantly higher in m. vastus lateralis following BR
supplementation. Taken together, these findings suggest that dietary nitrate supplementation
ACCEPTED
Copyright © 2013 by the American College of Sports Medicine. Unauthorized reproduction of this article is prohibited.
reduces muscle oxygen extraction which is consistent with the mechanisms proposed by both
Bailey et al. (2) and Larsen et al. (26) as discussed previously.
Engan et al. (13) found that BR ingestion increased SpO2 during a static apnea hold and
increased maximal apneic duration by approximately 11%. The authors suggest that the
substantial reduction in SpO2 during a maximal apnea may, as previously described, be partly
offset by an increased availability of NO. In direct contrast, a similar study by Schiffer et al. (35)
reported that dietary nitrate supplementation actually reduced both SpO2 and breath hold
duration during a static apnea. However, the authors also assessed the effects of BR
supplementation on an apnea during light intensity exercise (50 W). With this experimental
protocol there was a trend towards higher SpO2 during maximum effort apnea in the BR trial
than the PLA trial. Comparative analysis is difficult due to the profound differences in
methodologies employed by the two studies including the breath hold training status of the
participants, the supplementation strategy, the inhalation procedure prior to apnea and the
placement of the probe to measure SpO2. Despite this, a reduction in SpO2 during a static apnea
following BR supplementation may not be entirely unexpected as the NO mediated vasodilation
in the microcirculation would enhance peripheral blood perfusion, augmenting arterial oxygen
desaturation (35). The contrasting findings during the exercise apnea may be a consequence of
the working skeletal muscles becoming the dominant consumer of oxygen during exercise with
nitrate supplementation reducing the rate of oxygen extraction as previously described.
Therefore, there is good evidence from our study and others (28,35) that dietary nitrate
supplementation results in a small increase in SpO2 during exercise in hypoxia.
Despite the physiological and ergogenic effects demonstrated here, a single dose of BR has
recently been suggested to be less effective than a chronic (~6 days) supplementation protocol
ACCEPTED
Copyright © 2013 by the American College of Sports Medicine. Unauthorized reproduction of this article is prohibited.
(5,9,30,31,40). Recent evidence supporting the use of more prolonged supplementation has been
reported by Hernandez et al. (18) who demonstrated that 7 days of BR ingestion increased force
production of the fast twitch muscle fibers in mice which was associated with an alteration in
muscle protein expression. Furthermore, the reported improvement in muscle blood flow (16)
and in vitro mitochondrial phosphate to oxygen ratio (26) occurred following a more prolonged
period of nitrate supplementation (5 and 3 days respectively). It could be argued that the lack of
performance effects in these studies may be attributed to the use of highly trained or elite
endurance athletes in contrast to the trained or recreationally active participants employed in this
and other studies. Identifying why this may be the case is problematic because of differences in
supplementation protocols and exercise modalities. It should, however, be emphasized that the
baseline nitrate/nitrite pool is higher in endurance trained athletes than in untrained matched
controls, which may partially explain these differences (34).
Recent studies have also shown that individual variability in the response to dietary nitrate
supplementation may influence the subsequent impact on exercise performance. Data presented
by Wilkerson et al. (40) suggests there may be a responder vs. non-responder phenomenon with
dietary nitrate supplementation. They report a correlation between the change in plasma nitrite
and the change in exercise performance following nitrate supplementation and define a
“responder” as an increase in plasma nitrite of > 30% following nitrate supplementation. When
examining the TT and nitrite data from individual participants, the current study would seem to
support this hypothesis. Despite a mean increase of 134% in plasma nitrite, similar to Lansley et
al. (24) (138%) but substantially greater than others (4,40) (16 and 25% respectively), the change
in plasma nitrite levels in two of our nine participants would place them in the non-responder
category (Fig. 2). One of these participants completed the TT slower in the BR condition
ACCEPTED
Copyright © 2013 by the American College of Sports Medicine. Unauthorized reproduction of this article is prohibited.
compared to the PLA condition (the only one of the nine) and the other improved his
performance by just 0.2%. Nevertheless, we did not find that the differences in plasma nitrite and
TT performance between BR and PLA conditions across the cohort of cyclists were significantly
correlated. Nor indeed was there a correlation between baseline plasma nitrite concentration and
TT performance in either the PLA or BR trials. It is worth noting that one participant appears to
be an outlier that may be a consequence of the well described inter-individual variability in the
response to normobaric hypoxia (17). When this participant was removed from the analysis, the
correlation between the change in plasma nitrite and TT performance between PLA and BR
conditions was improved (R = –0.601, P = 0.115). The individual variability in the response to
nitrate supplementation is unquestionably a key issue and further research investigating the
impact of training status, baseline nitrite concentration, and environmental conditions is
recommended.
It is acknowledged that a limitation of the current study was that the consumption of nitrate rich
foods in the days preceding each test was not controlled and the use of antibacterial mouthwash
was not restricted. Despite this, the increase in plasma nitrite following ingestion of BR in the
present study is among the largest in the published literature to date yet no changes in nitrite or
nitrate were observed following PLA. A further limitation that should be acknowledged is that
the normobaric conditions of the exercise trials do not truly represent the hypobaric hypoxia at
true altitude. However, while further research in this area is clearly warranted, it is likely that our
findings would hold true under hypobaric hypoxic conditions as the PO2 is the critical factor
limiting exercise performance at altitude (28).
ACCEPTED
Copyright © 2013 by the American College of Sports Medicine. Unauthorized reproduction of this article is prohibited.
CONCLUSION
The principal findings of the present study were that a single dose of BR three hours prior to
exercise at a simulated altitude of 2500m resulted in a substantial reduction in VO 2 and a small
increase in SpO2 during submaximal exercise that was coupled with an improvement in a 16.1
km TT performance. Although we have reported that BR is a practical yet effective ergogenic aid
for exercise at simulated altitude, additional work is required to investigate the mechanism
responsible for this effect and the optimum supplementation strategy in order to maximize
performance.
ACKNOWLEDGMENTS
The authors thank all the participants who volunteered for this study. The authors disclose that
no funding was received for this work and have no conflicts of interest to declare. The results of
the present study do not constitute an endorsement by the American College of Sports Medicine.
ACCEPTED
Copyright © 2013 by the American College of Sports Medicine. Unauthorized reproduction of this article is prohibited.
REFERENCES
1. Bailey SJ, Winyard P, Vanhatalo A, et al. Dietary nitrate supplementation reduces the O2
cost of low intensity exercise and enhances tolerances to high intensity exercise in
humans. J Appl Physiol. 2009;107:1144–1155.
2. Bailey SJ, Fulford J, Vanhatalo A, et al. Dietary nitrate supplementation enhances muscle
contractile efficiency during knee-extensor exercise in humans. J Appl Physiol.
2010;109:135–48.
3. Beall CM, Laskowski D, Strohl KP, et al. Pulmonary nitric oxide in mountain dwellers.
Nature. 2001;414:411–412.
4. Bescos R, Rodriguez FA, Iglesias X, Ferrer MD, Iborra E, Pons A. Acute administration
of inorganic nitrate reduces VO2peak in endurance athletes. Med Sci Sports Exerc.
2011;43:1979–1986.
5. Bescos R, Ferrer-Roca V, Galilea PA, et al. Sodium Nitrate Supplementation Does Not
Enhance Performance of Endurance Athletes. Med Sci Sports Exerc. 2012;44(12), 2400–
2409.
6. Bond H, Morton L, Braakhuis AJ. Dietary nitrate supplementation improves rowing
performance in well-trained rowers. Int J Sport Nutr Exerc Metab. 2012;22:251–256.
7. Castello PR, David PS, McClure T, Crook Z, Poyton RO. Mitochondrial cytochrome
oxidase produces nitric oxide under hypoxic conditions: Implications for oxygen sensing
and hypoxic signaling in eukaryotes. Cell Metab. 2006;3:277–287.
8. Cermak NM, Gibala MJ, van Loon LJ. Nitrate supplementation’s improvement of 10-km
time-trial performance in trained cyclists. Int J Sport Nutr Exerc Metab. 2012;22(1): 64–
71.
ACCEPTED
Copyright © 2013 by the American College of Sports Medicine. Unauthorized reproduction of this article is prohibited.
9. Cermak NM, Res P, Stinkens R, Lundberg JO, Gibala MJ, van Loon L JC. No
Improvement in Endurance Performance Following a Single Dose of Beetroot Juice. Int J
Sport Nutr Exerc Metab. 2012;22(6), 470–478.
10. Droma Y, Hanaoka M, Ota M, et al. Positive association of the endothelial nitric oxide
synthase gene polymorphisms with high-altitude pulmonary edema. Circulation.
2002;106:826–830.
11. Duplain H, Sartori C, Lepori M, et al. Exhaled nitric oxide in high-altitude pulmonary
edema: role in the regulation of pulmonary vascular tone and evidence for a role against
inflammation. Am J Respir Crit Care Med. 2000;162:221–224.
12. Easton C, Howe CCF, Matzko RO, Piaser F, Pitsiladis YP. Impact of K4b2 calibration
drift on respiratory measurements during walking and running. Med Sci Sports Exerc.
2012;44(5):S346.
13. Engan HK, Jones AM, Ehrenberg F, Schagatay E. Acute dietary nitrate supplementation
improves dry static apnea performance. Respir Physiol Neurobiol. 2012;182:53–59.
14. Erzurum SC, Ghosh S, Janocha AJ, et al. Higher blood flow and circulating NO products
offset high-altitude hypoxia among Tibetans. Proc Natl Acad Sci USA. 2007;104:17593–
17598.
15. Faiss R, Pialoux V, Sartori C, Faes C, Dériaz O, Millet GP. Ventilation, Oxidative Stress,
and Nitric Oxide in Hypobaric versus Normobaric Hypoxia. Med Sci Sports Exerc,
2013;45(2):253-260.
16. Ferguson SK, Hirai DM, Copp SW, et al. Impact of dietary nitrate supplementation via
beetroot juice on exercising muscle vascular control in rats. J Physiol. 2013;591.2:547-
557.
ACCEPTED
Copyright © 2013 by the American College of Sports Medicine. Unauthorized reproduction of this article is prohibited.
17. Ge RL, Witkowski S, Zhang Y, et al. Determinants of erythropoietin release
in response to short-term hypobaric hypoxia. J Appl Physiol. 2002;92:2361–2367.
18. Hernández A, Schiffer TA, Ivarsson N, et al. Dietary nitrate increases increases tetanic
[Ca2+]I and contractile force in mouse fast-twitch fibres. J Physiol. 2012;590:3575–83.
19. Hoit BD, Dalton ND, Erzurum SC, et al. Nitric oxide and cardiopulmonary
hemodynamics in Tibetan highlanders. J Appl Physiol. 2005;99:1796–1801.
20. Heunks LM, Cody MJ, Geiger PC, Dekhuijzen PN, Sieck GC. Nitric oxide impairs Ca2+_
activation and slows cross-bridge cycling kinetics in skeletal muscle. J Appl Physiol.
2001;91: 2233–2239.
21. Jeukendrup A, Craig NP, Hawley JA. The bioenergetics of world class cycling. J Sci
Med Sport. 2000;3:414–433.
22. Kapil V, Milsom AB, Okorie M, et al. Inorganic nitrate supplementation lowers blood
pressure in humans. Hypertension. 2010;56: 274–281.
23. Lansley K, Winyard PG, Fulford J, et al. Dietary nitrate supplementation reduces the O2
cost of walking and running: a placebo-controlled study. J Appl Physiol. 2011;110:591–
600.
24. Lansley KE, Winyard PG, Bailey SJ, et al. Acute dietary nitrate supplementation
improves cycling time trial performance. Med Sci Sports Exerc. 2011;43:1125–1131.
25. Larsen FJ, Ekbolm B, Sahlin K, Lundberg JO, Weitzberg E et al. Effects of dietary nitrate
on oxygen cost during exercise. Acta Physiol. 2007;191:59–66.
26. Larsen FJ, Schiffer TA, Borniquel S, et al. Dietary inorganic nitrate improves
mitochondrial efficiency in humans. Cell Metab. 2011;13(2):149–159.
27. Lundberg JO, Weitzberg E, Gladwin M. The nitrate–nitrite–nitric oxide pathway in
ACCEPTED
Copyright © 2013 by the American College of Sports Medicine. Unauthorized reproduction of this article is prohibited.
physiology and therapeutics. Nat Rev Drug Discov. 2008;7:156–67.
28. Masschelein E, Van Thienen R, Wang X, Van Schepdael A, Thomis M, Hespel P.
Dietary nitrate improves muscle but not cerebral oxygenation status during exercise in
hypoxia. J Appl Physiol. 2012;113:736–745.
29. Modin A, Bjorne H, Herulf M, Alving K, Weitzberg E, Lundberg JO. Nitrite-derived
nitric oxide: a possible mediator of ‘acidic-metabolic’ vasodilation. Acta Physiol Scand.
2001;171(1):9–16.
30. Muggeridge DJ, Howe CCF, Spendiff O, Pedlar C, James PE, Easton C. The Effects of a
Single Dose of Concentrated Beetroot Juice on Performance in Trained Flatwater
Kayakers. Int J Sport Nutr Exerc Metab. 2013: in Press Available from:
http://journals.humankinetics.com/ijsnem-in-press/ijsnem-in-press/the-effects-of-a-
single-dose-of-concentrated-beetroot-juice-on-performance-in-trained-flatwater-kayakers
31. Peacock O, Tjonna AE, James P, et al. Dietary Nitrate Does Not Enhance Running
Performance in Elite Cross-country Skiers. Med Sci Sports Exerc. 2012;44(11):2213–
2219.
32. Rogers SC, Khalatbari A, Gapper PW, Frenneaux MP, James PE. Detection of human red
blood cell-bound nitric oxide. J Biol Chem. 2005;280: 26720–26728.
33. Rolfe DF, Hulbert AJ, Brand MD. Characteristics of mitochondrial proton leak and
control of oxidative phosphorylation in the major oxygenconsuming tissues of the rat.
Biochim. Biophys. Acta 1994;1188:405–416.
34. Schena F, Cuzzolin L, Rossi L, Pasetto M, Benoni G. Plasma nitrite/nitrate and
erythropoietin levels in cross-country skiers during altitude training. J Sports Med Phys
Fitness, 2002;42(2):129–134.
ACCEPTED
Copyright © 2013 by the American College of Sports Medicine. Unauthorized reproduction of this article is prohibited.
35. Schiffer TA, Larsen FJ, Lundberg JO, Weitzberg E, Lindholm P. Effects of dietary nitrate
on static and dynamic breath-holding on humans. Respir Physiol Neurobiol.
2013;185(2):339-348
36. Stamler JS, Meissner G. Physiology of nitric oxide in skeletal muscle. Physiol Rev.
2001;81:209–237.
37. van Beekvelt MC, Collier WN, Wevers RA, van Englan BG. Performance of near-
infrared spectroscopy in measuring local O(2) consumption and blood flow in skeletal
muscle. J Appl Physiol. 2001;90:511-519.
38. Vanhatalo A, Fulford J, Bailey SJ, Blackwell JR, Winyard PG, Jones AM. Dietary nitrate
reduces muscle metabolic perturbation and inproves exercise tolerance in hypoxia. J
Physiol. 2011;589:5517–5528.
39. Webb AJ, Patel N, Loukogeorgakis S, et al. Acute blood pressure lowering,
vasoprotective, and antiplatelet properties of dietary nitrate via bioconversion to nitrite.
Hypertension. 2008;51:784–790.
40. Wilkerson DP, Hayward GM, Bailey SJ, Vanhatalo A, Blackwell JR, Jones AM.
Influence of acute dietary nitrate supplementation on 50 mile time trial performance in
well-trained cyclists. Eur J Appl Physiol. 2012;112(12):4127–4134.
ACCEPTED
Copyright © 2013 by the American College of Sports Medicine. Unauthorized reproduction of this article is prohibited.
Figure Legends
Fig. 1 Plasma nitrate concentration 3 h post supplementation during PLA and BR trials. Data are
presented as individual responses (dashed lines) and mean (solid line) ± S.E.M. (error bars); *
denotes significant difference from PLA – BR supplementation (P < 0.001)
Fig. 2 Plasma nitrite concentration 3 h post supplementation during PLA and BR trials. Data are
presented as individual responses (dashed lines) and mean (solid line) ± S.E.M. (error bars); *
denotes significant difference from PLA – BR supplementation (P = 0.004)
Fig. 3 Oxygen consumption during submaximal exercise following PLA (white circles) and BR
(black circles) supplementation. Data are presented as the mean ± S.E.M. (error bars); * denotes
significant difference between PLA and BR (P = 0.049).
Fig. 4 Peripheral oxygen saturation during submaximal exercise following PLA (white circles)
and BR (black circles) supplementation. Data are presented as the mean ± S.E.M. (error bars).
Fig. 5 Time to complete 16.1 km TT following PLA and BR supplementation. Data are
presented as individual times (dashed line) and the mean time (solid line) ± S.E.M. (error bars). *
denotes significant difference between PLA and BR (P = 0.006).
ACCEPTED
Copyright © 2013 by the American College of Sports Medicine. Unauthorized reproduction of this article is prohibited.
Figure 1
ACCEPTED
Copyright © 2013 by the American College of Sports Medicine. Unauthorized reproduction of this article is prohibited.
Figure 2
ACCEPTED
Copyright © 2013 by the American College of Sports Medicine. Unauthorized reproduction of this article is prohibited.
Figure 3
ACCEPTED
Copyright © 2013 by the American College of Sports Medicine. Unauthorized reproduction of this article is prohibited.
Figure 4
ACCEPTED
Copyright © 2013 by the American College of Sports Medicine. Unauthorized reproduction of this article is prohibited.
Figure 5
ACCEPTED
Copyright © 2013 by the American College of Sports Medicine. Unauthorized reproduction of this article is prohibited.
