Acute and chronic effects of dietary nitrate supplementation on blood pressure
and the physiological responses to moderate-intensity and incremental
Anni Vanhatalo,1Stephen J. Bailey,1Jamie R. Blackwell,1Fred J. DiMenna,1Toby G. Pavey,2
Daryl P. Wilkerson,1Nigel Benjamin,2Paul G. Winyard,2and Andrew M. Jones1
1School of Sport and Health Sciences and2Peninsula College of Medicine and Dentistry, St. Luke’s Campus,
University of Exeter, Heavitree Road, Exeter, United Kingdom
Submitted 23 March 2010; accepted in final form 4 August 2010
Vanhatalo A, Bailey SJ, Blackwell JR, DiMenna FJ, Pavey TG,
Wilkerson DP, Benjamin N, Winyard PG, Jones AM. Acute and
chronic effects of dietary nitrate supplementation on blood pressure and
the physiological responses to moderate-intensity and incremental exer-
cise. Am J Physiol Regul Integr Comp Physiol 299: R1121–R1131, 2010.
First published August 11, 2010; doi:10.1152/ajpregu.00206.2010.—
Dietary nitrate (NO3
4–6 days has been shown to reduce the O2 cost of submaximal
exercise and to improve exercise tolerance. However, it is not known
whether shorter (or longer) periods of supplementation have similar
(or greater) effects. We therefore investigated the effects of acute and
physiological responses to moderate-intensity exercise and ramp in-
cremental cycle exercise in eight healthy subjects. Following baseline
tests, the subjects were assigned in a balanced crossover design to
receive BR (0.5 l/day; 5.2 mmol of NO3
l/day low-calorie juice cordial) treatments. The exercise protocol (two
moderate-intensity step tests followed by a ramp test) was repeated
2.5 h following first ingestion (0.5 liter) and after 5 and 15 days of BR
and PL. Plasma nitrite concentration (baseline: 454 ? 81 nM) was
significantly elevated (?39% at 2.5 h postingestion; ?25% at 5 days;
?46% at 15 days; P ? 0.05) and systolic and diastolic BP (baseline:
127 ? 6 and 72 ? 5 mmHg, respectively) were reduced by ?4%
throughout the BR supplementation period (P ? 0.05). Compared
with PL, the steady-state V˙O2during moderate exercise was reduced
by ?4% after 2.5 h and remained similarly reduced after 5 and 15
days of BR (P ? 0.05). The ramp test peak power and the work rate
at the gas exchange threshold (baseline: 322 ? 67 W and 89 ? 15 W,
respectively) were elevated after 15 days of BR (331 ? 68 W and
105 ? 28 W; P ? 0.05) but not PL (323 ? 68 W and 84 ? 18 W).
These results indicate that dietary NO3
BP and the O2 cost of submaximal exercise and that these effects are
maintained for at least 15 days if supplementation is continued.
?) supplementation with beetroot juice (BR) over
?supplementation on resting blood pressure (BP) and the
?/day) and placebo (PL; 0.5
?supplementation acutely reduces
beetroot juice; O2uptake; ramp exercise; performance; nitric oxide
IT IS WIDELY RECOGNIZED THAT a diet rich in vegetables is
beneficial for human health and is associated with a long life
span (44). Nitrate (NO3
is particularly abundant in leafy greens and beetroot, has
emerged as a possible mediating component for the cardiovas-
cular health benefits associated with high vegetable consump-
tion, such as is typical for the Mediterranean diet (13, 18, 32).
Dietary nitrate is reduced to bioactive nitrite NO2
tive anaerobic bacteria in the saliva and further to nitric oxide
(NO) via various pathways (7, 14, 50). NO has numerous
?), which is found in all vegetables and
functions in the body, including the regulation of blood flow,
muscle contractility, myocyte differentiation, glucose and cal-
cium homeostasis, and mitochondrial respiration and biogene-
sis (11, 13, 41).
Recent investigations suggest that the therapeutic potential
of increased NO bioavailability may extend beyond the well-
known hemodynamic effects (1, 2, 27, 28). Specifically, it has
been shown that dietary supplementation with sodium nitrate
(0.1 mmol·kg?1·d?1) resulted in a significant reduction in
pulmonary oxygen uptake (V˙O2) during submaximal exercise
(28). The use of pharmacological sodium nitrate is regulated in
many countries as a result of some earlier reports of a link
between foods containing nitrate and nitrite (via conversion to
N-nitrosamines) and cancer in laboratory animals (e.g., 34).
This restriction has been questioned by more recent findings
elucidating the benefits of nitrate-rich fruits and vegetables in
the diet (reviewed in Refs. 16 and 32), and it is known that
vegetables such as beetroot provide a natural and healthy
source of nitrate, which is widely available. We recently
demonstrated that 4–6 days of dietary supplementation with
nitrate-rich beetroot juice (BR; 0.5 l/day, equivalent to ?6
mmol/day of NO3
plasma nitrite concentration ([NO2
exercise at the same submaximal work rate (i.e., improved
exercise efficiency) and resulted in an extended time to ex-
haustion during high-intensity exercise (1). The results of these
studies are striking, particularly when considering that the
V˙O2-work rate relationship during submaximal exercise is
considered to be largely independent of factors such as age,
health status, aerobic fitness, and training status (22, 33).
In previous studies, the nitrate supplementation protocol
has involved around 3 days of supplementation (?0.07–
0.10 mmol·kg?1·day?1of NO3
with restrictions placed on habitual dietary nitrate intake in
both the placebo and experimental conditions (1, 27, 28).
However, although 4–6 days of NO3
in beneficial effects on blood pressure (BP) and the O2cost of
moderate exercise compared with a reduced NO3
dition, it is presently not known whether these same effects are
evident following shorter or longer supplementation periods. If
improvements in exercise efficiency were evident following
acute (i.e., single bolus) NO3
have important implications for performance enhancement.
Also, it is possible that continued dietary nitrate supplementa-
tion (i.e., beyond 6 days) might result in further improvements
in exercise efficiency. It is also important to investigate
whether the effects of dietary nitrate supplementation on ex-
?), which resulted in a significant increase in
?]), reduced the O2cost of
?) prior to any exercise testing
?supplementation, this would
Address for reprint requests and other correspondence: A. M. Jones, School
of Sport and Health Sciences, St. Luke’s Campus, Univ. of Exeter, Heavitree
Road, Exeter, EX1 2LU, UK (e-mail: email@example.com).
Am J Physiol Regul Integr Comp Physiol 299: R1121–R1131, 2010.
First published August 11, 2010; doi:10.1152/ajpregu.00206.2010.
0363-6119/10 Copyright © 2010 the American Physiological Society http://www.ajpregu.orgR1121
ercise efficiency are still present when habitual dietary nitrate
intake is not restricted during the control condition.
In a recent study, Webb et al. (45) assessed the effects of a
single dose of BR on plasma [NO2
remained close to this peak value until 5 h postingestion, and
returned to baseline after 24 h (45). The systolic and diastolic
BP and the mean arterial pressure (MAP) were significantly
reduced 2.5 to 3 h after BR intake, and the change in systolic
BP was temporally linked to plasma [NO2
cost of submaximal exercise is similarly linked to plasma
after BR ingestion. There are currently no published reports on
the effects of continued, daily nitrate supplementation on
ciency beyond 6 days. Interestingly, sustained exposure of
mammalian cells to NO over 6 days has been shown to induce
mitochondrial biogenesis through cGMP-dependent pathways
(10, 35). An increase in mitochondrial content might be man-
ifested at the systemic level as an increased maximal oxygen
uptake (V˙O2max) and/or gas exchange threshold (GET; analo-
gous with the lactate threshold) (5). Such effects would not be
expected following a single nitrate dose, but may be detectable
after prolonged supplementation.
The purpose of this study was to investigate the acute (2.5 h)
and chronic (up to 15 days) effects of dietary nitrate supple-
mentation on the O2cost of moderate-intensity constant-work-
rate cycle exercise, and on the GET, V˙O2max, and peak work
rate measured during a ramp incremental test to exhaustion.
The following hypotheses were tested: 1) that the plasma
after one 0.5-liter dose of BR and after 5 and 15 days of
supplementation at 0.5 l/day; 2) that the O2cost of moderate-
intensity exercise would be reduced 2.5 h after the first dose
and after 5 and 15 days of supplementation; and 3) that the
V˙O2maxand the GET would not be affected 2.5 h after the first
dose but would be increased after 5 and 15 days of supple-
?] and BP over 24 h. Plasma
?] reached a peak concentration at 3 h postingestion,
?]. If the reduced O2
?], this effect should also be detectable within 2.5 to 3 h
?] and related effects on BP and exercise effi-
?] would be elevated, and the BP would be reduced 2.5 h
Subjects. Eight healthy participants (including 3 females) volun-
teered for the study (means ? SD: age 29 ? 6 yr, body mass 71.8 ?
11.5 kg, height 1.75 ? 0.05 m). Prior to testing, subjects were
informed of the protocol and the possible risks and benefits of
participation before written informed consent was obtained. All pro-
cedures were approved by the institutional research ethics committee
and were conducted in accordance with the Declaration of Helsinki.
Subjects were physically active but not highly trained in any particular
sport. Subjects were instructed to adhere to their normal exercise
routine and diet throughout the experimentation. The subjects kept a
physical activity and dietary diary and were asked to perform similar
activities and consume similar meals in the first and second supple-
mentation periods. Prior to data collection, subjects were fully famil-
iarized with exercise testing and performed a preliminary ramp incre-
mental test (which was not included in the data analyses) to minimize
any possible learning effect. Subjects were instructed to avoid stren-
uous exercise for 24 h prior to each testing session. Exercise tests were
conducted at the same time of the day ? 2 h for each subject. Subjects
were asked to arrive into the laboratory adequately hydrated and
having refrained from consuming alcohol for 24 h and food or
caffeine for 3 h before each test.
Presupplementation tests. Subjects made two visits to the labora-
tory prior to the commencement of dietary supplementation. All
exercise tests were performed on an electrically braked cycle ergome-
ter (Lode Excalibur Sport, Groningen, The Netherlands). During the
first visit, a ramp incremental cycling test was performed for the
assessment of the GET and V˙O2max. Subjects pedaled at a self-selected
constant cadence (80 rpm, n ? 3; 70 rpm, n ? 5). The cadence and
saddle and handlebar settings were replicated for all subsequent tests.
Following 3 min of unloaded pedaling, the work rate was increased by
1 W every 2 s (i.e., 30 W/min) until the subject reached volitional
exhaustion. The test was terminated when the cadence fell by ? 10
rpm below the chosen cadence, despite strong verbal encouragement.
The power output reached at the point of exhaustion was recorded as
the peak power output. All timing devices were covered from the test
subject and no feedback was given on the elapsed time or power
output during the test. Feedback on performance was only given once
all experimentation for the entire study had been completed. Pulmo-
nary gas exchange was measured breath by breath and recorded
throughout the test.
During the second visit, subjects performed two-step transitions
from a 20-W baseline to moderate-intensity cycling at 90% GET.
Each bout lasted for 5 min, and bouts were separated by 10 min of
rest. Pulmonary V˙O2 was measured breath by breath, and the data
from the two bouts were time aligned and averaged to improve the
signal-to-noise ratio (26). This test provided a baseline measurement
of the V˙O2dynamics and the O2cost (?V˙O2/?work-rate) of moderate
exercise. During both presupplementation visits, the resting BP was
measured, and a resting venous blood sample was taken for the
NO availability (13, 19, 25, 29). The mean values of all presupple-
mentation measurements were used as presupplementation baseline
for BP variables and plasma [NO2
Supplementation period. Following completion of the presupple-
mentation tests, subjects were assigned using a balanced, randomized
crossover design to receive organic BR (0.5 l/day containing ?5.2
mmol of NO3
placebo (PL; low-calorie blackcurrant juice cordial with negligible
supplementations with the 15-day supplementation periods separated
by a 10-day washout period (Fig. 1A). Four subjects started with BR,
and the other four started with PL. Throughout the study period,
subjects were instructed to maintain their normal daily activities and
food intake, i.e., unlike in previous studies (1, 2, 27, 28) where
subjects were not instructed to minimize consumption of nitrate-rich
foods. The subjects were not aware of the experimental hypotheses to
be tested but were informed that the purpose of the study was to
compare the physiological responses to exercise following the con-
sumption of two commercially available sports beverages. This study
was completed before the publication of our earlier studies (1, 2), such
that the subjects were not aware that BR might be ergogenic.
On day 1 of the supplementation period, subjects provided a venous
blood sample for the measurement of plasma [NO2
BP was measured. The subjects then consumed a 0.5-liter dose of the
supplement over a 15-min period and rested for 2.5 h. During this time
subjects were allowed to drink water, but no other beverages or food
were ingested. Two and a half hours after the ingestion of the
supplement a second blood sample was obtained, BP was measured,
and the exercise test was commenced. The test protocol consisted of
two 5-min bouts of moderate-intensity cycling and a ramp incremental
test to exhaustion (as described for the presupplementation tests), with
all bouts separated by 10-min recovery (Fig. 1). Subjects were then
prescribed a dose of 0.5 l/day of supplement (BR or PL) and instructed
to consume it in two equal doses in the morning and in the evening.
Subjects returned to the laboratory on days 2, 5, 8, 12, and 15 for the
measurement of plasma [NO2
supplement intake had occurred 2.5–5 h prior to the start of testing.
The exercise protocol (Fig. 1) was also repeated on days 5 and 15.
?, Beet It; James White Drinks, Ipswich, UK) and
?content). Thus, all eight subjects underwent both BR and PL
?], and the resting
?] and BP, ensuring that the latest
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