Efeitos do Suco de Beterraba em Exercício

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Acute effects of various doses of nitrate-rich beetroot juice on 
high-intensity interval exercise responses in women: 
a randomized, double-blinded, placebo-controlled, crossover 
trial
Zhang Jiaqia, Dai Zihana, Stephen Heung-Sang Wong a, Zheng Chen a,b 
and Eric Tsz-Chun Poon a
aThe Chinese University of Hong Kong, Department of Sports Science and Physical Education, Hong Kong, 
Shatin, New Territories, China; bThe Educational University of Hong Kong, Department of Health and 
Physical Education, Hong Kong, Tai Po, New Territories, China
ABSTRACT
Background: This study investigated the acute effects of various 
doses of nitrate-rich beetroot juice on the responses to high- 
intensity interval exercise in women.
Methods: A double-blinded, randomized, placebo-controlled, 
crossover trial was conducted with 13 recreationally active young 
women (age = 23 ± 2 years). All participants performed interval 
exercise (8 × 1-min bouts of cycling at 85% of peak power output 
[PPO] interspersed with 1-min active recovery at 20% of PPO) 2.5 h 
after consumption of the randomly assigned beetroot juice contain-
ing 0 mmol (placebo), 6.45 mmol (single-dose), or 12.9 mmol (dou-
ble-dose) NO�3 . The heart rate (HR), blood pressure, blood lactate, 
blood glucose, oxygen saturation, rating of perceived exertion 
(RPE), and emotional arousal were assessed.
Results: Nitrate supplementation significantly altered the HR and 
RPE responses across the three trials. The mean HR was lower in the 
single- and double-dose groups than in the placebo control group 
during both work intervals and recovery periods, as well as across 
the overall protocol (all pmass between men and women may affect 
impact the storage, utilization, and retention of nitrates following supplementation. 
Moreover, sex-associated difference in resting blood pressure, as indicated by lower 
resting blood pressure in young women than in men of similar age, is speculated to be 
related to estrogen levels [45]. This disparity in baseline values, may result in different 
lowering effect of nitrate supplementation on blood pressure. Despite these potential sex 
differences, women remain underrepresented as research participants in this research 
field. Therefore, it is crucial to include women in investigations on the effects of nitrate- 
rich beetroot juice during exercise.
Given that the majority of existing literature has primarily focused on endurance-based 
exercise protocols, applied various nitrate doses, and exhibited a predominant interest in 
men, the current study aimed to investigate the acute dose–response effects of nitrate 
supplementation by utilizing a high-intensity interval exercise protocol and recruiting 
women participants. We hypothesized that a higher dose (12.9 mmol) of acute NO�3 intake 
would induce greater physiological responses as indicated by HR, blood pressure, blood 
glucose, lactate, and oxygen saturation, as well as psychological responses as indicated by 
RPE and emotional arousal during high-intensity interval cycling than a lower dose (6.45 
mmol) and placebo control (0 mmol).
2. Materials and methods
2.1. Participants
Thirteen healthy, recreationally active women with a regular menstrual cycle 
(age = 22.9 ± 1.8 years, body mass = 56.4 ± 6.4 kg, BMI = 21.1 ± 1.9 kg/m2, and peak 
power output [PPO] = 123 ± 25 W) enrolled voluntarily in this study. Participants were 
included if they 1) had a BMI between 18.5 and 24.9 kg/m2 [46] and 2) met the require-
ment of moderate level of physical activity per week (i.e. equivalent to 150 min of 
moderate intensity physical activity performed per week), as assessed by the 
International Physical Activity Questionnaire [47]. Participants were excluded if they 1) 
had blood pressure ≥130/80 mmHg; 2) took medication that affects vasodilation, heart 
rate, or stomach acid production; 3) took anti-coagulant medications; 4) took oral contra-
ceptive pills; 5) had orthopedic limitations; 6) had a diagnosis of cardiovascular disease or 
Type I or II diabetes; or 7) had a history of myocardial infarction, uncompensated heart 
failure, or unstable angina pectoris [48,49].
During the screening process, the participants were informed of the experimental 
procedures, provided written, informed consent, and completed health history and 
physical activity questionnaires. The study protocol adhered to and respected the tenets 
of the Declaration of Helsinki and was approved by the Survey and Behavioral Research 
224 Z. JIAQI ET AL.
Ethics Committee of the Chinese University of Hong Kong (Reference No. SBRE-21-0511. 
Date of approval: 15 February 2022).
2.2. Experimental design
This was a randomized, double-blind, placebo-controlled, crossover trial. After 
recruitment, the participants reported to the laboratory on four separate occasions 
over 11 days. Visit 1 was a preliminary test for anthropometric measurements and 
the PPO test. During visits 2, 3, and 4, the participants arrived at the laboratory in 
a rested state (abstinence from strenuous exercise 24 h preceding the trial) in the 
morning after an 8-h overnight fast and performed interval exercise 2.5 h after the 
consumption of the randomly assigned dose of beetroot juice with a standard 
meal. A washout period of 72 h was used to separate each visit to allow for 
sufficient recovery, in line with previous research investigating the acute effects 
of beetroot juice supplementation [50,51]. A maximum washout period of up to 6 
days was allowed under special circumstances (e.g. illness). All trials were per-
formed at the same time of day (±1 h) and under controlled environmental condi-
tions (20ºC–22ºC and 30–36% humidity). Participants were asked to record their 
food intake 24 h prior to the first main trial and replicate the same diet in the 24 h 
preceding all subsequent trials. They were also required to refrain from eating 
foods or drinks with caffeine or a high nitrate content 24 h prior to each trial, as 
well as antibacterial mouthwash and chewing gum for the entire study duration, as 
they would attenuate the reduction of nitrate to nitrite in the oral cavity [52]. 
Furthermore, the participants were asked to report any gastrointestinal intolerance 
or side effects from consuming the supplementation at the end of the last experi-
mental trial. The overall study design is illustrated in Figure 1.
Figure 1. Overview of the experimental protocol and methodological aspects of study timeline. 
Double = double-dose group; Single = single-dose group; Placebo = placebo control group; IPAQ, 
International Physical Activity Questionnaire.
JOURNAL OF THE INTERNATIONAL SOCIETY OF SPORTS NUTRITION 225
2.3. Supplementation procedures
The nitrate-rich beetroot juice (batch number: 1K30) and placebo beverage (batch num-
ber: 2A07) employed in the present study were commercially available and purchased 
from Beet It Sport, James White Drinks Ltd. (Suffolk, England, United Kingdom). The 
company consistently provided nitrate content within the manufacturer’s specifications 
and normally tested random batches to a third-party testing service provider for nitrates 
levels. Both beetroot juices were made with the same ingredients: 98% concentrated 
beetroot juice plus 2% concentrated lemon juice. The nutrients in the placebo shots were 
the same as the standard ones with the only difference in nitrate concentrations. In 
placebo beverages, nitrates are extracted using an ion-exchange resin while maintaining 
the same taste, appearance (color), and macronutrients. A previous study examining the 
nitrate content of beetroot juice and placebo beverages confirmed the product integrity 
[53]. To ensure blinding, the nitrate-rich beetroot juice and placebo beverage were 
packaged with number labels by and independent research student.
During visits 2, 3, and 4, the participants arrived at the laboratory under fasting 
conditions. They randomly consumed either 2 × 70 mL nitrate-rich beetroot juice (double- 
dose group, ~12.9 mmol NO�3 ), 70 mL nitrate-rich beetroot juice +70 mL placebo bever-
age (single-dose group, ~6.45 mmol NO�3 ), or 2 × 70 mL placebo beverage (placebo 
control group, ~0.0068 mmol NO�3 ) along with a standard meal (one portion of scrambled 
egg, one chicken steak, and one piece of toast). The total caloric intake of the meal 
(including the beverage) was 771.2 kcal, which consisted of 42.2 g of carbohydrates, 46 
g of fat, and 42.2 g of protein. The caloric value is calculated by a registered dietitian using 
a local government food database (https://www.cfs.gov.hk/english/nutrient/fc- 
introduction.php). During the postprandial period, the participants rested in the lab for 
2.5 h and were allowed to consume only plain water.
2.4. PPO test
During visit 1, all participants completed an incremental exercise test on an ergometer 
bike (LC7, Monark, Sweden) at a constant, self-selected pedal rate (60–80 rpm). The seat 
height was appropriately adjusted, recorded, and reproduced in all subsequent trials. 
Initially, participants performed 3 min of baseline cycling at 40 W. Thereafter, the work 
rate increased at a rate of 20 W·min−1 until reaching the limit of tolerance. The test was 
completed when the pedal rate fell below 55 rpm despite verbal encouragement. The 
power output achieved at the point of exhaustion was recorded as the PPO. Blood glucose 
and lactate levels were measured at baseline and exhaustion respectively, and the HR was 
monitored throughout the test.
2.5. Interval cycling exercise protocol
The trial began 2.5 h after the consumption of beetroot juice to allow the timing to 
coincide with peak nitrate/nitrite bioavailability[54]. The participants warmed up for 5 
min (cycling at 40 W at a self-selected pedal rate). Then, they performed the interval 
exercise (8 × 1-min bouts of cycling at 85% of PPO interspersed with 1-min active recovery 
at 20% of PPO), followed by 5-min cool-down cycling at 40 W at the same pedal rate. 
226 Z. JIAQI ET AL.
https://www.cfs.gov.hk/english/nutrient/fc-introduction.php
https://www.cfs.gov.hk/english/nutrient/fc-introduction.php
Given that this approach to interval training represents a midpoint with respect to 
intensity, recovery, and total volume, it is considered to be a “medium-volume” high- 
intensity interval exercise that is more appropriate for the general population [55]. All 
work intervals were preceded by a 3-s countdown with a “GO” command. Verbal encour-
agement and information pertaining to the interval number were provided during inter-
mittent exercise.
2.6. Assessments of physiological responses
HR was monitored continuously through a heart rate sensor (Polar Team2 System, Polar 
Electro, Finland). Blood pressure was measured upon arrival (after a 15-min rest period in 
a seated position), pre-interval exercise, and post-interval exercise using an automatic 
sphygmomanometer (M7 Intelli IT, Omron, Japan). Blood glucose and lactate concentra-
tions were measured using capillary blood samples from the fingertips with portable 
analyzers (Contour Plus Glucometer, Bayer Healthcare, Germany; Lactate Meter, Nova 
Biomedical Co., USA). Blood oxygen saturation (SpO2) was measured using a sensor 
placed on the fingertip (Yuwell YX306, Yuwell, China). Measurements of glucose, lactate, 
and SpO2 were taken four times, during the last 15 s of warm-up, within the first 15 s of 
recovery period 4, within the first 15 s of recovery period 8, and during the last 15 s of 
cool-down.
2.7. Assessments of psychological responses
Participants indicated their rating of perceived exertion (RPE) at the end of each stage 
using a 20-grade RPE scale (Borg scale) [56]. The validated scale was printed, and 
participants were able to visualize the scale when required.
Emotional arousal was measured before and after the exercise to describe the senti-
ments experienced during the trial using the 5-point Self-Assessment Manikin (SAM). This 
questionnaire used cartoons to reflect emotional experiences in terms of valence (posi-
tivity or negativity of emotion), arousal (intensity of emotion), and dominance (control 
over emotion) [57].
2.8. Sample size calculation
Sample size calculation was performed using G * POWER software (Heinrich-Heine- 
Universitӓt Düsseldorf, Germany) with an alpha of 0.05, a statistical power of 0.8 and 
a Cohen’s f value of 0.5 based on previous literature related to the effect of nitrate 
consumption on HR during interval exercise [39]. Accordingly, at least nine participants 
were required for the study. A plausible drop-out rate was set at 20%; therefore, 13 
participants were recruited for this study.
2.9. Statistical analysis
All data were analyzed using SPSS software package (IBM SPSS version 26.0, 
Chicago, IL, USA). All continuous variables were presented as means ± SDs. The 
normality of the data was checked before further analysis. Data collected during 
JOURNAL OF THE INTERNATIONAL SOCIETY OF SPORTS NUTRITION 227
the preliminary test and data for HR and RPE variables during the work intervals, 
recovery periods, and across the overall protocol in the main trials were analyzed 
using one-way analysis of variance (ANOVA). The Huynh – Feldt correction was 
applied when Mauchly’s sphericity test reached significance (p ≤ 0.05). The remain-
ing data collected during the main trials were analyzed using a two-way (treat-
ment × time) repeated-measures ANOVA to explore the effect of interventions 
(double-dose, single-dose, and placebo) over time on the magnitude of each 
dependent variable. A Bonferroni post hoc comparison was performed when 
ANOVA significance was reached. Partial eta squared (η2
p) was used to indicate 
the magnitude of the difference among trials. Scores of 0.01, 0.06, and >0.14 were 
considered small, moderate, and large effect sizes, respectively [58]. Statistical 
significance was set at p ≤ 0.05.
3. Results
All participants completed three interval exercise trials and complied with the supple-
mentation protocol. None of the participants withdrew from the study because of adverse 
responses to the nitrate supplementation or the exercise protocol. No illness was reported 
during the trial. A CONSORT flow diagram of study recruitment is shown in Figure 2.
3.1. Peak power output test
During the preliminary test, HR, RPE, blood glucose, and lactate levels at exhaustion were 
significantly different from those at baseline (Table 1). HR at exhaustion was 175 ± 10 beats/ 
min, compared to 121 ± 18 beats/min at baseline (F[2.06, 24.68] = 177.99, pthere was no interaction effect between treatment and time on either SBP 
(F [4,70] = 0.88, p = .480, η2
p = .048) or DBP (F [4,70] = 0.84, p = .503, η2
p = .046).
The effects of different doses of nitrate supplementation on blood glucose and lactate 
concentrations are shown in Table 2. ANOVA analyses revealed that there was neither 
a main effect by time (F[1.77, 63.76] = 2.42, p = .104, η2
p = .063) nor an interaction effect 
between treatment and time on blood glucose (F[3.54, 63.76] = 1.08, p = .370, η2
p = .057). 
In contrast, there was a main effect by time on blood lactate concentration 
(F[2.43, 87.54] = 135.14, pbenefits 
observed with the beetroot juice containing 12.9 mmol NO�3 .
Consistent with a previous study [39], the mean HR was lower during intermittent 
exercise following nitrate supplementation than in the control. However, in contrast to 
Table 2. (Continued).
Placebo Single Double p-value
Arousal pre-exercise 4 ± 1 4 ± 1 4 ± 1 .651
Arousal post-exercise 3 ± 1 3 ± 1 3 ± 1 .701
Dominance pre-exercise 3 ± 1 4 ± 1 4 ± 1 .909
Dominance post-exercise 4 ± 1 4 ± 1 3 ± 1 .054
HR = heart rate; RPE = rating of perceived exertion; SBP = systolic blood pressure; DBP = diastolic blood pressure; SpO2 = 
blood oxygen saturation; Placebo = placebo control group; Single = single-dose group; Double = double-dose group. 
Data are presented as mean ± standard deviation; p-values of the Levene’s test for equality of variances are reported.
232 Z. JIAQI ET AL.
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athletic performance. In 
addition, since the participants in our study were recreationally active young women, who 
do not look for an improvement in performance during competition, hence, we measured 
outcomes that were closer to real life setting (e.g. perceived exertion, emotional arousal). 
The underlying motivation was to figure out whether nitrate supplementation would 
lower people’s perceived discomfort and become more engaged/aroused during exer-
cise, as traditional high-intensity exercise is hypothesized to have a lower exercise 
adherence due to the greater displeasure caused by higher intensity exercise above the 
ventilatory threshold [65].
Our study contributes to the limited body of research investigating the effects of nitrate 
supplementation in women. Furthermore, the present study provides new insights into the 
efficacy of nitrate supplementation in alleviating perceived effort during interval exercise, 
highlighting the practical benefits of this supplement. However, our study has a few limita-
tions. Our sample size calculation was based on the primary outcome of the HR effect, which 
may have resulted in insufficient statistical power to detect the dose–response relationship 
on some secondary outcomes due to the relatively small sample size. Another limitation is 
the lack of measurement of plasma nitrate and nitrite concentrations following acute con-
sumption of beetroot juice. Future studies should consider larger sample sizes to detect 
potential differences between groups and examine how fluctuations in plasma or muscle 
nitrate and nitrite concentrations would affect the physiological responses to interval exer-
cise. Furthermore, only oral contraceptives intake was included as the exclusion criteria in the 
present study. Although none of the participants in our study had used any form of contra-
ceptives, it is encouraged that future studies should consider excluding women applying 
other forms of contraceptives, for example, contraceptive patches, that control endogenous 
sex hormones concentrations as oral contraceptives. Finally, during postprandial resting 
period, although participants were only allowed to consume plain water, we did not control 
for the amount of water intake. Future study should consider recording and monitoring the 
water intake to ensure the consistency of water consumption among participants.
JOURNAL OF THE INTERNATIONAL SOCIETY OF SPORTS NUTRITION 235
5. Conclusions
In conclusion, our study demonstrated that acute nitrate ingestion in the form of beetroot 
juice significantly decreased the mean HR and RPE during work intervals, recovery periods, 
and across the overall protocol in women. However, additional benefits for these para-
meters were not evident after a higher dose of acute beetroot juice supplementation.
Acknowledgments
We would like to thank all the participants involved in this study. We also acknowledge DZH. for the 
involvement on the preparation of the blinded packages.
Disclosure statement
No potential conflict of interest was reported by the author(s).
Funding
The author(s) reported there is no funding associated with the work featured in this article.
ORCID
Stephen Heung-Sang Wong http://orcid.org/0000-0002-6821-4545
Zheng Chen http://orcid.org/0000-0003-0453-3341
Eric Tsz-Chun Poon http://orcid.org/0000-0002-0842-1323
Authors’ contributions
The study and methodology were conceived using EP and ZJQ. ZJQ and DZH were involved in data 
collection and data treatment. Writing was conducted by ZJQ. All authors reviewed critically the 
manuscript under SW supervision.
Data availability statement
Datasets used in this study are available from the corresponding author under reasonable request.
Publication statement
This manuscript has not been published elsewhere.
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	Abstract
	1. Introduction
	2. Materials and methods
	2.1. Participants
	2.2. Experimental design
	2.3. Supplementation procedures
	2.4. PPO test
	2.5. Interval cycling exercise protocol
	2.6. Assessments of physiological responses
	2.7. Assessments of psychological responses
	2.8. Sample size calculation
	2.9. Statistical analysis
	3. Results
	3.1. Peak power output test
	3.2. Effects of beetroot juice on interval cycling responses
	3.2.1. Assessments of physiological responses
	3.2.2. Assessments of psychological responses
	4. Discussion
	5. Conclusions
	Acknowledgments
	Disclosure statement
	Funding
	ORCID
	Authors’ contributions
	Data availability statement
	Publication statement
	References