nutrients

Article
Beetroot Juice Produces Changes in Heart Rate Variability and
Reduces Internal Load during Resistance Training in Men: A
Randomized Double-Blind Crossover
Jose Manuel Jurado-Castro 1,2,3, * , David Casanova-Rodriguez 4 , Julian Campos-Perez 5 ,
Francisco Jesus Llorente-Cantarero 2,6 , Candelaria Alonso De La Florida-Villagran 4 ,
Víctor Manuel Diaz-Bernier 4 and Antonio Ranchal-Sanchez 4,7, *

1 Metabolism and Investigation Unit, Maimonides Biomedical Research Institute of Cordoba (IMIBIC),
Reina Sofia University Hospital, University of Cordoba, 14004 Cordoba, Spain
2 CIBER Fisiopatología de la Obesidad y Nutrición (CIBEROBN), Instituto de Salud Carlos III,
28029 Madrid, Spain
3 Ciencias De La Actividad Física y El Deporte, Escuela Universitaria de Osuna (Centro Adscrito a la
Universidad de Sevilla), 41640 Osuna, Spain
4 Department of Nursing, Pharmacology and Physiotherapy, Faculty of Medicine and Nursing,
University of Cordoba, 14071 Cordoba, Spain
5 Department of Food Science and Technology, Campus Universitario de Rabanales, University of Cordoba,
14004 Cordoba, Spain
6 Department of Specific Didactics, Faculty of Education, University of Cordoba, 14004 Cordoba, Spain
7 Grupo De Investigación Clínico Epidemiológica De Atención Primaria, Maimonides Biomedical Research
Institute of Cordoba (IMIBIC), Reina Sofia University Hospital, University of Cordoba, 14004 Cordoba, Spain
* Correspondence: juradox@gmail.com (J.M.J.-C.); en1rasaa@uco.es (A.R.-S.)
Citation: Jurado-Castro, J.M.;
Casanova-Rodriguez, D.;
Campos-Perez, J.; Llorente-Cantarero, Abstract: Beetroot juice (BJ) has been used as a sport supplement, improving performance in resis-
F.J.; De La Florida-Villagran, C.A.; tance training (RT). However, its effect on the modulation of the autonomic nervous system has not
Diaz-Bernier, V.M.; Ranchal-Sanchez, yet been widely studied. Therefore, the objective of this randomized double-blind crossover study
A. Beetroot Juice Produces Changes was to assess the effect of acute BJ supplementation compared to placebo in blood pressure (BP),
in Heart Rate Variability and Reduces heart rate (HR), heart rate variability (HRV) and internal load during RT measure as Root Mean
Internal Load during Resistance Square of the Successive Differences between adjacent RR intervals Slope (RMSSD and RMSSD-Slope,
Training in Men: A Randomized
respectively). Eleven men performed an incremental RT test (three sets at 60%, 70% and 80% of their
Double-Blind Crossover. Nutrients
repetition maximum) composed by back squat and bench press with. HR, HRV and RMSSD-Slope
2022, 14, 5119. https://doi.org/
were measured during and post exercise. As the main results, RMSSD during exercise decrease
10.3390/nu14235119
in the BJ group compared to placebo (p = 0.023; ES = 0.999), there were no differences in RMSSD
Academic Editor: Robert Wessells post-exercise, and there were differences in RMSSD-Slope between groups in favor of the BJ group
Received: 6 November 2022
(p = 0.025; ES = 1.104) with a lower internal load. In conclusion, BJ supplementation seems to be a
Accepted: 26 November 2022 valuable tool for the reduction in the internal load of exercise during RT measured as RMSSD-Slope
Published: 2 December 2022 while enhancing performance.

Publisher’s Note: MDPI stays neutral

Keywords: heartbeat; heart rate control; dietary supplement; nitric oxide; autonomic nervous system;
with regard to jurisdictional claims in
beta vulgaris
published maps and institutional affil-
iations.

1. Introduction
Copyright: © 2022 by the authors. Beetroot juice (BJ) and its influence on sport performance has been studied [1], showing
Licensee MDPI, Basel, Switzerland. a great interest in the sport nutrition field and the sport performance in different disciplines,
This article is an open access article and not least, being also interesting on general population health reducing different cardio-
distributed under the terms and vascular and coronary diseases [1,2]. Mainly, the effects of BJ and its interest on the field
conditions of the Creative Commons
of health, sport nutrition and sport performance are mostly based by the principal active
Attribution (CC BY) license (https://
principle present of BJ supplementation, which is the dietary inorganic nitrate (NO3 − ) [3].
creativecommons.org/licenses/by/
After its ingestion, the NO3 − goes through a complex digestion and convoluted metabolic
4.0/).

Nutrients 2022, 14, 5119. https://doi.org/10.3390/nu14235119 https://www.mdpi.com/journal/nutrients

Nutrients 2022, 14, 5119 2 of 12

pathways. It is first absorbed by the salivary circulation and subsequently reduced to nitrite
(NO2 ) by the action of nitrate reductase facultative anaerobic bacteria present at the dorsal
surface of the tongue [4]. Then, at the stomach, NO2 is decomposed into nitric oxide (NO)
and finally reaches the plasma and systemic circulation [5,6].
In sport performance, the physiological effects of BJ supplementation and NO could
be summed up in a dilation of the vascular endothelium [7,8], causing a vasodilator effect
and reducing the blood pressure (BP) [9,10]. In addition, these physiological effects are
interesting for sport performance because it could increase muscle blood flow [11], alter and
improve lactate removal in the exercise [12]. In addition, the intake of NO3 − effects has been
proved in different endurance sports, where cardiovascular system has an important role
in performance [1,7,13], showing a lower oxygen consumption (VO2) during exercise [1]
with an improvement in adenosine triphosphate (ATP) synthesis [14].
Nevertheless, in strength sports or resistance training (RT), BJ supplements have
received less attention [3]. However, research has shown BJ as an effective ergogenic aid
in RT, observing an increase in muscle strength, explosive force and muscular endurance
due to better ATP utilization, increased blood volume, and therefore, oxygen during
exercise [15–19].
In the cardiovascular system, NO regulates various functions such as contractile
force, myocardial relaxation, mitochondrial respiration and coronary perfusion [20], and
it clearly has a cardioprotective role in pathologies such as myocardial infarction and
heart failure [21]. In animal models of cardiac ischemia–reperfusion, treatment with NO2
produced a clear cardioprotective effect [22–25]. However, the results of human studies are
not so obvious [26].
Normally, a healthy heart is not a regular metronome with the same interval between
heartbeats, but it experiences variations of the normal rhythm of the heart. To measure
and control these different oscillations of the period between consecutive heartbeats, heart
rate variability (HRV) is a proved valuable tool [27]. HRV can be used as a mirror of the
cardiorespiratory control system and parasympathetic function of the autonomic nervous
system (ANS), being a useful signal for understanding the status of the ANS [28]. According
to a meta-analysis [29], HRV data obtained in men and women cannot be treated equally
and that studies need to characterize these differences. In autonomic control of the heart
indexed by HRV measures, it has been observed that women showed a significantly lower
mean RR interval, showing greater vagal activity [30].
HRV has a direct relationship between sport performance and physical activity and
their physiological effects, decreasing with stress activities such as exercise and when
respiratory increases [31]. Recently, HRV has been considered useful to determine the
internal load of physical activity [32], evaluating the modulation of the sympathetic and
parasympathetic system, specifically, to know the activation of the parasympathetic system
in the athlete’s recovery [33–36]. Using HRV, certain data obtained in his measurement,
such as the Root Mean Square of the Successive Differences between adjacent RR intervals
(RMSSD), could be one of the best reliable measures of parasympathetic activity. A mea-
surement in a short period of time is sufficient [37]. In this way, Naranjo Orellana et al. have
determined the measure of internal load based on the recovery of the RMSSD-Slope on
30 min post-exercise to monitor the effect of workloads and fatigue caused by exercise [32].
Although an electrocardiogram, measured by measurement instruments such as the
Holter system, would be the reference method for assessing HRV [38], it not suitable
for daily measurement and sport. In this regard, new technologies such as wearable de-
vices [39–42] and smartphone applications [43,44] have taken the spotlight, becoming the
reference method for measuring RR intervals. Chest straps such as the Polar H10 (Polar Inc.,
Kempele, Finland) can be considered the gold standard for RR interval assessments if in-
tense activities with strong body movements are investigated. Polar H10 has demonstrated
the validity of for the detection of RR intervals in a wide range of physical activities and
sports [39]. HRV parameters and several nonlinear parameters can be further interpreted
and analyzed using advanced HRV analysis software such as Kubios HRV (University of
Nutrients 2022, 14, 5119 3 of 12

Eastern Finland, Kuopio, Finland) [45]. This software supports several input data formats
for electrocardiogram data and beat-to-beat RR interval data.
Despite the knowledge of the ergogenic component of BJ and the changes produced in
BP after its consumption pre-exercise, certain physiological post-exercise recovery effects
have not been evaluated during RT, especially in relation to HR and HRV, which is a
critical period in which various modifications occur, including changes in autonomic
modulation [46], which can promote an environment conducive to the development of
abnormal alteration in both BP, HR and HRV [46,47]. Thus, due the possible effects of BJ
supplementation in addition to RT in cardiovascular system, the purpose of this study was
to investigate the possible effects of BJ acute supplementation during RT in BP and the
variations in HR, HRV and internal load during RT measured by changes in HRV in man.
We hypothesized that the BJ consumption could produce changes in the HRV and produce
a reduction in the internal load during RT.

2. Materials and Methods

2.1. Design
The study was conducted according to the CONSORT statement (Supplemental Material S1)
with a double-blind, randomized crossover trial design. The experimental procedure was carried
out in 3 visits, with a difference of one week between visits. Body composition was assessed
at the first visit in addition to a familiarization protocol and 1RM tests. In the second and
third visits, the participants carried out the experimental supplementation protocol (BJ
consumption or placebo). In order to standardize the influence of the circadian rhythm,
and the possible variation of muscle strength and power [48,49], the visits took place at the
same time of day in the morning (±0.5 h) with a temperature of 24 ◦ C ± 1 ◦ C.

2.2. Participants
The inclusion/exclusion criteria to recruit trained men were: (a) 18–30 years old;
(b) experience of more than 2 years in RT; (c) familiarization with the back squat and bench
press exercises; (d) abstinence from the consumption of nutritional supplements or anabolic
substances for three months before the study; and (e) absence of musculoskeletal injuries.
These criteria were verified through personal interviews.
All participants were informed through an information sheet and a signed written con-
sent before the start of the investigation in accordance with the Declaration of Helsinki [50].
The protocol was approved by Portal de Ética de la Investigación Biomédica de Andalucía
ethics committee (protocol code: BEETROOT JUICE; reference 4284).

2.3. Anthropometry and Body Composition

During the first visit, anthropometric measurements were made according to the
protocol of the International Society for the Advancement of Kinanthropometry (ISAK).
Body composition was reported using bioelectrical impedance (MC-780MA; Tanita) and
height was recorded with a stadiometer (portable stadiometer; Seca 214) [51].

2.4. Study Interventions

2.4.1. Familiarization Protocol, One-Repetition Maximum Testing and Resistance Training
Performance (Back Squat and Bench Press)
Before the test, participants performed a familiarization by performing a maximum
explosive speed in the concentric movement for the back squat and bench press exercises.
The participants lifted 20 kg on a Smith machine (Technogym, Barcelona, Spain) for a
total of three repetitions controlling the technique movement of back squat. They received
correction by research if necessary.
During the familiarization and 1RM test, the execution velocity was controlled using a
linear position transducer (v.4.1, Speed4Lift; Madrid, Spain) used in previous studies [52,53].
A previous testing protocol to find the 1RM load in back squat and bench press was
realized [54].
Nutrients 2022, 14, 5119 4 of 12

Back squat and bench press were performed on the same day, and the protocol was
replicated for both. Test specifications were published previously by Ranchal-Sanchez et al.
(2020) [52].
Results regarding performance in back squat and bench press as well as RPE have
been published by Ranchal-Sanchez et al. (2020), finding an overall better performance in
the RT with the BJ consumption, improving the muscular resistance without differences in
ratings of perceived exertion (RPE) [52].

2.4.2. Supplementation Protocol

First, 70 ml of BJ (BEET It Sport® ; James White Drinks Ltd., Ipswich, UK) with
6.4 mmol·L−1 or 400 mg NO3 − per serving [55] or 70 mL of blackcurrant drink without
NO3 − as a placebo (Capri-Sun, Uxbridge, UK) were taken 120 min before of each visit [1].
Participants completed a 24 h dietary recall, on the day prior to the first visit, as a tool
for athletes to replicate their diet [17]. Moreover, they also received nutritional guidelines
based on the exchange of food groups to guarantee that 48 h before each visit, they followed
a similar diet composed of 60% carbohydrates, 30% lipids and 10% proteins [14,56–58], and
a list of foods rich in NO3 − (e.g., beetroot, celery, or spinach) or rich in caffeine that they
could not consume.
Twenty-four hours before the experimental visits, the participants were instructed to
refrain from brushing their teeth and the use of mouthwash, and the participants had to
sleep at least 8 h to ensure optimal hydration and rest during the study period.

2.5. Study Outcomes

2.5.1. Blood Pressure
For testing blood pressure, diastolic (DBP) and systolic (SBP) blood pressure in the
non-dominant arm were measured (OMROM, HEM-7200-E2 device) at the beginning and
the RT post-exercise, measuring three times to dismiss possible measurement failures [59].

2.5.2. Heart Rate and Heart Rate Variability Measurement

During training and recovery, HR was monitored with a Polar H10 heart rate monitor.
As the data receiver, the Polar Beat application was used in dual Bluetooth connection to
record HR (HR average and maximum HR of the session), allowing a second connection to
be made to measure HRV, on another mobile device with the Elite HRV application, which
was previously validated for HRV recording [60].
HRV measurement was performed by recording the last 5 min of training and after
finishing it for a further 10 min in a relaxed sitting position [32]. Due to the sudden
change in HRV caused by the transition between training and recovery, the first 5 min of
recovery were discarded [43], finally obtaining the values of 5 min at post-exercise for later
interpretation.
The RR time intervals obtained from the HRV recording were later downloaded and
analyzed using the Kubios HRV software (Version 3.3, University of Eastern Finland,
Kuopio, Finland) [61]. Each record was previously analyzed to detect the possible presence
of noise and abnormal beats, applying the corresponding filters if necessary.
During HRV measurement, RMSSD was recorded during exercise and at post-exercise.
These data were later used to calculate the RMSSD-Slope by using the following formula as
indicated by Naranjo-Orellena et al. (2019) [32]:

RMSSD-SLope = (RMSSD-postexercise − RMSSD-exercise)/time exercise

For an intensity of 60–75% of the maximum effort (coinciding with the intervals of the
present study, 60–80%), the internal load score proposal according to the RMSSD-Slope
value was: poor (<0.4); acceptable (0.4–2.6); very good (>2.6) [32].
Nutrients 2022, 14, 5119 5 of 12

2.5.3. Sample Size

The sample size was calculated considering recent studies on the effects of BJ in
RT [16,17]. A calculation based on a normal distribution was performed, with a power of
0.80 and a 2-tailed α level set to 0.05; the minimum number of participants required was
estimated as 11.

2.5.4. Randomization
A third person from outside the research team randomized all participants’ supple-
ments (50% of participants took BJ and 50% took placebo at each visit). The online program
https://www.randomlists.com/team-generator (accessed on 2 February 2020) was used.

2.6. Statistical Analysis

To assess the normality of the variables, Shapiro–Wilk tests were performed, and the
equality of variance was contrasted with the Levene test. The comparison of the mean
outcomes between BJ or placebo consumption was realized with a paired-samples t-test.
For a practical significance of the results, effect size (ES) was calculated using Hedges g for
repeated measures. ESs were considered to have large (ES > 0.8), moderate (ES = 0.8–0.5),
small (ES = 0.5–0.2), or trivial (ES < 0.2) effects [62]. In addition, a general linear model for
repeated measures was applied for the time–supplement interaction effect for the SBP, DBP,
RMSSD first and final measurements. The Greenhouse–Geisser adjustment for sphericity
was calculated. After a significant F-test, differences among the means were identified
using pairwise comparisons with Bonferroni’s adjustment. A general linear model for
repeated measures ES were calculated using partial eta squared (η 2 p ), considering small
to be under 0.25, medium 0.26–0.63, and large above 0.63 [63]. Significance was set at
p-value < 0.05. SPSS software (Version 22.0, IBM SPSS Statistics for Windows, 2013; IBM
Corp., Armonk, NY, USA) was used for the statistical analysis. The data are presented as
mean ± SD.

3. Results
Eleven men completed the two visits of the study protocol; one participant did not
complete the study due to gastrointestinal problems. Figure 1 shows the flow diagram.
Eleven participants were finally randomized, whose characteristics and anthropometrics
data were published in a previous paper [52].
There were no differences in BP basal and post-exercise either in the HR mean or
HR post-exercise. However, we did observe a difference in HR max, which was higher
in the BJ group (Table 1). No differences were observed either in the interaction time x
supplementation basal and post-exercise in the SBP (p = 0.444; η 2 p = 0.054), or in DBP
(p = 0.642; η 2 p = 0.020).

Table 1. Systolic and diastolic blood pressure basal and post-exercise, and heart rate during resis-
tance exercise.

Variable Beetroot Juice Placebo p-Value ES

Systolic BP Basal (mmHg) 133 ± 22.2 129.5 ± 20.9 0.641 0.156
Systolic BP post-exercise (mmHg) 122 ± 11.1 124.7 ± 10.2 0.472 0.244
Diastolic BP basal (mmHg) 72.4 ± 11.7 76.7 ± 8.1 0.271 0.411
Diastolic BP post-exercise (mmHg) 68.8 ± 7.6 71.5 ± 11.6 0.285 0.265
HR mean (ppm) 129.3 ± 11.8 125.6 ± 12.5 0.208 0.293
HR post-exercise (ppm) 106.6 ± 12.5 101.8 ± 16.9 0.161 0.311
Maximum HR (ppm) 174.6 ± 9.1 168.5 ± 10.2 0.022 * 0.607
BP: blood pressure; ES: effect size; HR: heart rate. *: significant differences (p-value < 0.05).
 Nutrients 2022, 14, x FOR PEER REVIEW 6 of
Nutrients 2022, 14, 5119 6 of 12

Nutrients 2022, 14, x FOR PEER REVIEW 7 of 13

Figure 1. Flow diagram
Figure utilizing
1. Flow Consolidated
diagram Standards of Reporting
utilizing Consolidated StandardsTrials (CONSORT)
of Reporting guidelines.
Trials (CONSORT) guid
lines.
Differences were found in the RMSSD during exercise with a lower value in the
consumption
were ofThere
no differencesBJ vs. placebo
inwere (BJ: 12.9 ±RMSSD
the post-exercise
no differences 6.3BP
in vs.basal
placebo:
(BJ: 26.5 26.9 ± 18;
and ±post-exercise
16 vs. p =either
placebo:0.023; ES
27.7in = 0.999).
±the
28.7;
HR p mean
= or H
There ES
0.914; were no differences
= 0.050) (Figure in
2). the
Therepost-exercise
was an RMSSD
interaction (BJ:
time 26.5
x ± 16 vs. placebo:
supplementation 27.7
between
post-exercise. However, we did observe a difference in HR max, which was higher in th ± 28.7;
the
p = 0.914;
first ES =measurement
and final 0.050) (Figurein2).RMSSD
There was an interaction
(p = 0.041; timeTherefore,
η2pobserved
= 0.354). x supplementation
there werebetween
differ-
BJ group (Table 1). No differences were either in the
2 = 0.354). interaction time x suppl
the first
ences in theand final measurement
RMSSD-Slope (BJ: 3in± RMSSD
3 (very (p =
good)0.041;
vs. placebo:
η p 0.5 ±Therefore,
0.7 there were
(acceptable); p = (p = 0.64
mentation basal and post-exercise in the SBP (p = 0.444; η p = 0.054), or in DBP
2
differences
0.025; in the RMSSD-Slope (BJ: 3 ± 3 (very good) vs. placebo: 0.5 ± 0.7 (acceptable);
ES = η1.104).
2p = 0.020).
p = 0.025; ES = 1.104).
Table 1. Systolic and diastolic blood pressure basal and post-exercise, and heart rate during r
sistance exercise.

Variable Beetroot Juice Placebo p-Value ES

Systolic BP Basal
133 ± 22.2 129.5 ± 20.9 0.641 0.156
(mmHg)
Systolic BP post-exercise
122 ± 11.1 124.7 ± 10.2 0.472 0.244
(mmHg)
Diastolic BP basal
72.4 ± 11.7 76.7 ± 8.1 0.271 0.411
(mmHg)
Diastolic BP post-exer-
68.8 ± 7.6 71.5 ± 11.6 0.285 0.265
cise (mmHg)
HR mean (ppm) 129.3 ± 11.8 125.6 ± 12.5
Figure 2. (A) Acute effects of BJ consumption compared to placebo on RMSSD during exercise;
0.208 0.293
(B) Acute HR post-exercise
effects of BJ consumption(ppm)
compared106.6 ± 12.5
to placebo onplacebo 101.8
post-exercise ± 16.9BJ: beetroot
RMSSD. 0.161 0.311
Figure 2. (A) Acute effects of BJ consumption compared to on RMSSD during exercise;juice.
(B)
*: Significant
Acute Maximum
differences
effects of HR (ppm)
< 0.05). to placebo on post-exercise RMSSD. BJ: beetroot juice. **:
(p-valuecompared
BJ consumption 174.6 ± 9.1 168.5 ± 10.2 0.022 0.607
BP: blood pressure;
Significant differences (p-Value <ES: effect size; HR: heart rate. *: significant differences (p-value < 0.05).
0.05).
4. Discussion
This study Differences
4. Discussion were found
aimed to investigate the in the RMSSD
possible effectsduring exercise
of acute with a lower value
BJ supplementation on BPin the co
sumption
and variations in HR,ofHRV,
BJ vs.and
placebo (BJ:load
internal 12.9 during
± 6.3 vs.RT.
placebo:
The 26.9 ±of
results 18;the
p =study
0.023;showed
ES = 0.999). The
This study aimed to investigate the possible effects of acute BJ supplementation on
thatand
BP BJ supplementation
variations in HR,before
HRV,training (120 min
and internal loadbefore)
duringcould
RT. produce
The resultschanges
of theinstudy
HRV,
showed that BJ supplementation before training (120 min before) could produce changes
in HRV, reducing RMSSD during exercise, but showing no changes in BP or RMSSD after
exercise. Therefore, it seems that BJ could reduce the internal load measured through the
RMSSD-Slope during RT, thus obtaining an improvement in RT performance, mainly in
Nutrients 2022, 14, 5119 7 of 12

reducing RMSSD during exercise, but showing no changes in BP or RMSSD after exercise.
Therefore, it seems that BJ could reduce the internal load measured through the RMSSD-
Slope during RT, thus obtaining an improvement in RT performance, mainly in muscular
endurance, as previously published [52].
Although there was no effect compared to placebo, BJ has the potential to decrease
BP, vascular resistance and myocardial oxygen demand in both recovering and exercising
subjects [64]. Vanhatalo et al. (2010) conclude that BJ reduces only SBP [65], whereas
Webb et al. (2008) indicated that reductions in both systolic and diastolic BP were observed,
respectively, 2.5 and 3 h post BJ supplementation [66]. Furthermore, systolic BP remained
decreased after 24 for hours post ingestion, while diastolic BP returned toward baseline [67].
As a whole, these data invite us to think that BJ or nitrate-rich supplementation diet is
more suitable to changes in systolic BP than diastolic. The result of this decline in BP can
attenuate the O2 cost of an increment in work rate by 20% especially the younger one is. In
fact, Stanaway et al. (2019) have described that SBP was reduced in young and older adults
following BR supplementation, while DBP was reduced only in the older ones [68].
A possible explanation for the results of the present study may be the reactive oxygen
and nitrate species (RONS) produced by RT [69]. Due to the antioxidant effect of NO3 −
and betalains [70–72], it could be theorized that the lack of effect in our study is due to
the oxidation of these components of the BJ carried out by the RONS produced during
the RT. There is some controversy in the mechanism by which BJ reduce BP, because
some reviews [9] and studies [65,68] suggest that this effect is due to NO3 − content, but
a recent systematic review and metanalysis carried out by Bahadoran et al. (2017) [73]
highlights that this effect could be NO3 − independent and may be related to other bioactive
components such as betalains or other antioxidants [74,75].
RT has been probed to decrease acute parasympathetic modulation regardless of
the age [76]. In the present study, it was shown how the BJ consumption modified the
maximum HR and decreased RT-intensive exercise-mediated RMSSD in trained young men,
reducing internal load compared with a control group. Similar to this, Benjamin et al. (2020)
found that beetroot extract accelerates the return of parasympathetic modulation during
recovery after an RT protocol in healthy adult men [77]. Although changes in HRV during
aerobic exercise have been studied, an increase in HRV was observed during the day [78,79].
Carrijo et al. (2021) conclude that a single dose of BJ, independent of NO3 − content, does
not change aerobic exercise-mediated responses in HRV indexes in time, frequency, and
non-linear domains in hypertensive postmenopausal women [80], which is aspect not
studied in trained men during RT.
In this regard, the BJ group had a significantly lower RMSSD during exercise than the
placebo group. Exercise enhances the activity of the sympathetic nervous system [81,82]
while reducing the vagal tone especially as the HR and exercise workload increase [83].
With RMSSD being a measurement of parasympathetic activation [84], besides the fact
that the BJ group accumulated a greater total number of repetitions [52] and achieved a
significantly higher HR than the placebo group, it could explain the reduced RMSSD during
exercise in this group. Anyway, there were no differences between groups in post-exercise
RMSSD, and the BJ group showed an increase in RMSSD-Slope between groups, which
would mean a decrease in the internal training load in the BJ group [32,37]. A possible
explanation for this could be the effect of NO on the autonomous nervous system, which
could inhibit the sympathetic activity while increasing the vagal outflow [85,86] and thus
enhance the recovery after exercise.
Despite the fact that, to our knowledge, no other clinical trials have studied the effects
of BJ during RT in HRV. The relationship between HRV, sport and the utilization of some
sport supplements has been analyzed before. In this regard, other commonly used sup-
plements such us creatine limits the parasympathetic modulation of the RT exercise [87],
whereas caffeine has contradictory results by increasing the parasympathetic modulation af-
ter anaerobic exercise [88] but delaying parasympathetic recovery after aerobic exercise [89].
Other not as common supplements such as the black thai ginger show a similar effect to
Nutrients 2022, 14, 5119 8 of 12

caffeine in the response of autonomic nervous system to anaerobic exercise [90]. In this
sense, studies that combine different supplements are required.
To our knowledge, this was the first study to evaluate the acute effects of BJ on HRV
and internal load during resistance training. The results should be interpreted with caution.
The main limitation of this study is the fact that only one measure of the HRV was realized
in both groups and, with HRV being a very variable between days, it can bias the results of
this trial. We suggest that future research on this topic should replicate HRV measurements
not only to know the acute effects but also chronic ones. Another possible limitation to the
study is the exercise order selection. Due to the back-squat exercise, which was performed
previous to the bench press, and because of the back-squat involving a larger muscle
volume [91,92], the accumulated neuromuscular fatigue and metabolic by-products [93]
could cause different interpretation of the results if HRV was measured after back-squat
instead of bench press.

5. Conclusions
In conclusion, no differences were found in BP between groups, but changes in HRV
were found. Specifically, a decrease in RMSSD during exercise was observed with BJ con-
sumption. It was accompanied with no differences in RMSSD post-exercise and an internal
load reduction measured by RMSSD-Slope after BJ consumption while performing better
on the muscular endurance test. Therefore, we conclude that BJ is a useful supplementation
tool increasing the parasympathetic regulation in RT and thus decreasing the internal load.

6. Practical Applications
HRV measurement is a practical tool that can give a broader physiological under-
standing of sports performance. The effects of certain nutritional substances and sports
supplements can affect the autonomic modulation of cardiovascular function. According
to the results of the present study on the reduction in internal load derived from HRV
parameters, it seems that BJ could be a suitable strategy to reduce exercise-derived fatigue
even with a higher volume of training during RT.

Supplementary Materials: The following supporting information can be downloaded at: https://
www.mdpi.com/article/10.3390/nu14235119/s1, Supplementary Material S1: checklist CONSORT.
Author Contributions: Conceptualization, J.M.J.-C.; methodology, J.M.J.-C. and A.R.-S.; software,
J.M.J.-C. and F.J.L.-C.; validation, J.M.J.-C. and A.R.-S.; formal analysis, J.M.J.-C.; investigation,
V.M.D.-B., C.A.D.L.F.-V. and J.M.J.-C.; resources, J.M.J.-C., A.R.-S. and J.C.-P.; data curation, J.M.J.-C.
and J.C.-P.; writing—original draft preparation, J.M.J.-C., D.C.-R. and A.R.-S.; writing—review and
editing, J.M.J.-C., D.C.-R., A.R.-S., V.M.D.-B., C.A.D.L.F.-V., F.J.L.-C. and J.C.-P.; visualization, J.M.J.-C.
and A.R.-S.; supervision, J.M.J.-C. and A.R.-S.; project administration, J.M.J.-C. and A.R.-S.; funding
acquisition, A.R.-S. All authors have read and agreed to the published version of the manuscript.
Funding: This research was supported by Departamento de Enfermería, Farmacología y Fisioterapia
(Facultad de Medicina y Enfermería, Universidad de Córdoba).
Institutional Review Board Statement: The study was conducted in accordance with the Declaration
of Helsinki, and approved by the Ethics Committee of Portal de Ética de la Investigación Biomédica
de Andalucía (protocol code: BEETROOT JUICE; reference 4284; April 2019).
Informed Consent Statement: Informed consent has been obtained from the patient(s) to publish
this paper.
Conflicts of Interest: The authors declare no conflict of interest.
Nutrients 2022, 14, 5119 9 of 12

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