Acute hydrogen-rich water ingestion stimulates cardiac autonomic activity in healthy females

Abstract

Background: Hydrogen-rich water (HRW) has been shown to have a stimulating effect on the human body. Objective: The aim of
the study was to assess the influence of acute HRW intake on autonomic cardiac regulation during 50 min of rest sitting. Methods:
Fourteen healthy females (age 21.7 ± 1.2 years, body mass 67.8 ± 8.7 kg, height 167 ± 5.5 cm) took part in this double-blind, randomized, placebo-controlled trial with crossover design. Heart rate variability (HRV) was monitored in the sitting position after administration of 1260 ml of HRW or placebo. Time domain indexes of HRV as the square root of the mean of the squares of differences
between adjacent RR intervals (RMSSD), the standard deviation of all RR intervals (SDNN) and the ratio of SDNN/RMSSD as an index
of sympatho-vagal balance were used to assess autonomic cardiac response. The values were transformed using natural logarithm
(Ln). Results: After administration of HRW, we found significantly increased ratio Ln SDNN/RMSSD when comparing it to placebo in
25 min (HRW: 0.40 ± 0.30, placebo: 0.26 ± 0.25, p = .049) and 35 min (HRW: 0.44 ± 0.30, placebo: 0.28 ± 0.28, p = .029) of rest
sitting. Ln SDNN was significantly increased after HRW administration when compared to placebo in 45 min (HRW: 4.41 ± 0.42 ms,
placebo: 4.28 ± 0.31 ms, p = .049) of rest sitting. Conclusions: Acute HRW ingestion induced a relative increase in sympathetic activity between 25 and 35 min post-ingestion, whereas vagal activity was not affected.

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reducing the biological reaction to radiation-induced oxi-
dative stress (Kang et al., 2011), other studies showed a
reduction of mitochondrial DNA damage (Tomofuji et al.,
2014) and improved autonomic cardiac function after 4
weeks of administration in healthy adults (Mizuno et al.,
2017). In addition to clinical studies, there is positive effect
also in physically active humans while HRW was reported
to reduce blood acidosis (Ostojic & Stojanovic, 2014) and
lactate concentration (Aoki et al., 2012; Botek et al., 2019,
2021), improve perceptual and ventilatory response to
exercise (Botek et al., 2019) or having an antifatigue effect
(Aoki et al., 2012; Botek et al., 2020, 2021; Da Ponte et al.,
2018). Zanini et al. (2020) found an alternative effect of
HRW to caffeine in terms of sleep deprivation where HRW
was suggested to have a stimulating effect on the brain, par-
ticularly improved sensory stimulation.
Autonomic nervous system (ANS) activity is com-
monly noninvasively assessed by heart rate variability
(HRV; Malik, 1997; Task Force of the European Society
of Cardiology, 1996). HRV reflects the complexity of the
physiological system and mirrors the time variations in the
subsequent heartbeats intervals which is primarily associ-
ated with respiratory related vagal activity (Acharya et al.,
2006). It has been reported that HRV analysis sensitively
* Corresponding author: Barbora Sládečková, e-mail barbora.sladeckova@upol.cz, ORCID® record https://orcid.org/0000-0001-6862-4571
Article history: Received November 25 2020, Accepted April 6 2021, Published April 27 2021
Copyright: © 2021 The Author(s). Published by Palacký University Olomouc. This is an open access article distributed under the terms of the Creative
Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are credited. This license does not cover any third-party material that may appear with permission
in the article.
ORIGINAL RESEARCH
Acute hydrogen-rich water ingestion stimulates cardiac autonomic
activity in healthy females
Michal Botek1, Barbora Sládečková1*, Jakub Krejčí1, František Pluháček2, and Eliška Najmanová2
1Department of Natural Sciences in Kinanthropology, Faculty of Physical Culture, Palacký University Olomouc, Olomouc, Czech
Republic; and 2Department of Optics, Faculty of Science, Palacký University Olomouc, Olomouc, Czech Republic
Abstract
Background: Hydrogen-rich water (HRW) has been shown to have a stimulating effect on the human body. Objective: The aim of
the study was to assess the influence of acute HRW intake on autonomic cardiac regulation during 50 min of rest sitting. Methods:
Fourteen healthy females (age 21.7 ± 1.2 years, body mass 67.8 ± 8.7 kg, height 167 ± 5.5 cm) took part in this double-blind, ran-
domized, placebo-controlled trial with crossover design. Heart rate variability (HRV) was monitored in the sitting position after admin-
istration of 1260 ml of HRW or placebo. Time domain indexes of HRV as the square root of the mean of the squares of differences
between adjacent RR intervals (RMSSD), the standard deviation of all RR intervals (SDNN) and the ratio of SDNN/RMSSD as an index
of sympatho-vagal balance were used to assess autonomic cardiac response. The values were transformed using natural logarithm
(Ln). Results: After administration of HRW, we found significantly increased ratio Ln SDNN/RMSSD when comparing it to placebo in
25 min (HRW: 0.40 ± 0.30, placebo: 0.26 ± 0.25, p = .049) and 35 min (HRW: 0.44 ± 0.30, placebo: 0.28 ± 0.28, p = .029) of rest
sitting. Ln SDNN was significantly increased after HRW administration when compared to placebo in 45 min (HRW: 4.41 ± 0.42 ms,
placebo: 4.28 ± 0.31 ms, p = .049) of rest sitting. Conclusions: Acute HRW ingestion induced a relative increase in sympathetic activ-
ity between 25 and 35 min post-ingestion, whereas vagal activity was not affected.
Keywords: molecular hydrogen, autonomic nervous system, heart rate variability, sympathetic activity, time domain indexes
ACTA GYMNICA, 2021, Volume 51, Article e2021.009
https://doi.org/10.5507/ag.2021.009
Introduction
Molecular hydrogen (H2) has been described as an anti-
inflammation agent (Ohta, 2015). Moreover, H2 was
reported as a strong selective antioxidant itself or gas able
to improve the activity of the antioxidant system showing
cytoprotective effect (Ohsawa et al., 2007). The antioxida-
tive effect of H2 was proved by neutralization of reactive
oxidative species and reactive nitrogen species (Nicolson et
al., 2016). The higher level of reactive oxidative and nitro-
gen species is associated with aging, daily stress, demand-
ing physical activity etc., where stressors can increase
sympathetic activity, and consequently the oxidative stress
(Steptoe et al., 2007). The human body could be exposed
to H2 in several ways as follows, inhalation of H2 (Fukuda
et al., 2007), intravenous infusion (Ishibashi et al., 2014)
of H2 or by more practical methods as intake of hydrogen
rich water (HRW; Kajiyama et al., 2008) and H2 bathing
(Kawamura et al., 2016). HRW is considered to be the easi-
est and the safest method of delivering H2 to the human
body (Nicolson et al., 2016). Clinical and animal studies
have demonstrated that HRW intake is linked to numerous
health benefits, such as improvement in lipid and glucose
metabolism in diabetes patients (Kajiyama et al., 2008),
quality of life after radiotherapy treatment in regards to
OPEN
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M. Botek et al. Acta Gymnica, 2021, 51, e2021.009
identifies even the tiny changes in autonomic cardiac altera-
tions that may occur with acute intake dose of water (Chen
et al., 2004).
A previous study has shown that 7.5 ml · kg–1 of water
intake elicited many cardiovascular changes, such as total
peripheral resistance, HRV and cardiovagal baroreflex sen-
sitivity, decrease in heart rate (HR), and a parallel increase
in HF component showing enhanced vagal activity (Brown
et al., 2005). Water drinking activates both the sympathetic
(Scott et al., 2001) and vagal branches (Routledge et al.,
2002) of the ANS. Physiological response to water drinking
in healthy subjects may be an integrated response including
increased sympathetic vasoconstrictor activity and at the
same time parallel increase of the vagal activity that pre-
vents a rise in arterial pressure by reducing cardiac output
(Jordan et al., 2000). This result corresponds to the study
of Chen et al. (2018) who found a rise in cardiac vagal
activity at 15 min post water intake. Similarly, Peçanha et
al. (2014) reporting faster cardiac vagal reactivation due to
water intake after high intensity exercise.
The present study aimed to investigate the effects of
acute HRW ingestion on autonomic cardiac response
in young healthy females during sitting at rest. In this
regard, we hypothesized that there would be a significant
increase in sympathetic activity and concurrent reduction
in vagal activity.
Methods
Participants
Fourteen healthy female university students (age 21.7 ± 1.2
years, body mass 67.8 ± 8.7 kg, height 167 ± 6 cm) took
part in this study. Prior to testing, all tested participants
were informed about the testing procedures and the aim of
the research. The day before testing, participants were not
exposed to any exhaustive activity. The participants were
advised to avoid drinking coffee, tea or any other substance
potentially affecting the ANS activity, for at least two hours
before the testing. The research was conducted in accor-
dance with the Declaration of Helsinki and was approved
by the Ethics Committee of the Faculty of Physical Cul-
ture, Palacký University Olomouc under reference number
75/2017. The participation of subjects in this research was
voluntary and all participants signed informed consent
with the testing procedure.
Anthropometric measures
The participants had their body height (cm) and body mass
(kg) measured using the SOEHNLE 7307 (Leifheit, Nas-
sau, Germany).
Experimental protocol
This is a double-blind, randomized, placebo-controlled
trial with a crossover design. The study protocol consisted
of 2 laboratory visits (Figure 1). The first visit included a
detailed explanation of the course of the experiment, the
signing of informed consent, body mass measurement, and
the random division of participants into two groups. Each
group contained 7 participants. Randomization was per-
formed by means of lots, using an equal number of strips
with two color (red and blue). Participants drew only one
strip whilst blinded. Then the experiment itself began.
Data for HRV analysis were measured in five segments
(Figure 1). Each segment lasted 5 min, as recommended
for standardized short-term recording (Task Force of the
Figure 1 Overview of the study protocol
Note. RR intervals were recorded in grey coloured segments. HRW = hydrogen rich water; Bas = baseline segment before any water administration; R15,
R25, R35, and R45 = recovery segments 15, 25, 35, and 45 min respectively after drinking all the water.

M. Botek et al. Acta Gymnica, 2021, 51, e2021.009
3
European Society of Cardiology, 1996). The baseline seg-
ment was measured before the administration of any water
and was used to calculate HRV reference values. Before the
baseline segment, there was a 5 min pause during which
the participants sat in a comfortable chair. This pause
served as a stabilization period to ensure RR intervals to
achieve stationarity.
After completing the measurement of the baseline seg-
ment, participants were asked to drink three packages of
beverage (HRW or placebo) within a 15 min time frame.
It was requested to drink one package every 5 min. After
15 min and drinking all the water, the recovery time began
to count. During the first 10 min, participants were sitting
and had the opportunity to visit the toilet to empty their
urinary bladder. This was followed by another 5 min stabi-
lization pause in which participants were sitting to achieve
stationary RR intervals. 15 min after drinking the last pack-
age, the recording of the first recovery segment labelled R15
was started. Participants were asked to sit without exces-
sive movement until the end of the entire experimental
protocol, during which three more recovery segments were
recorded. The segments started 25, 35, and 45 min after
drinking the last package and were labeled R25, R35, and
R45 respectively.
After 7 days of washout, a second visit took place, in
which beverages were exchanged (HRW for placebo and
vice versa).
HRW and placebo preparation
A total volume of 1260 ml HRW (Aquastamina HRW,
Nutristamina, Ostrava, Czech Republic) or placebo
(Aquastamina H2 free, Nutristamina, Ostrava, Czech
Republic) was administrated in three doses (420 ml each)
within 15 min. HRW and a placebo was served in identi-
cal packages so participants were not able to distinguish
between HRW and placebo because H2 is colorless, odor-
less and tasteless. HRW was produced by infusing H2
under high pressure directly into water. The characteris-
tic of both HRW and placebo (Table 1) were determined
using the pH/ORP/Temperature-meter (AD14, Adwa
Instruments, Szeged, Hungary) and H2Blue reagent (H2
Sciences, Henderson, NV, USA) according to the manu-
facturer instructions.
HRV monitoring
To calculate HR and HRV variables, RR intervals were
recorded using a Polar HR monitor (V800, Polar, Kem-
pele, Finland) with a resolution of 1 ms. All participants
wore watches and chest straps. Participants were allowed
to breathe spontaneously during the RR recording. Paced
breathing was not used because voluntary control of breath-
ing has been shown to affect HRV indexes (Patwardhan
et al., 1995). However, time domain analysis of HRV
is thought to be resistant to the effects of breathing fre-
quency compared to spectral analysis (Penttilä et al., 2001).
RR intervals were transferred to a computer using the
Polar Flow cloud service. Artifacts (ectopic beats, missing
beats, etc.) were identified by visual inspection and sim-
ply deleted because the deletion method provided the best
overall performance (Lippman et al., 1994). The square
root of the mean of the squares of differences between adja-
cent RR intervals (RMSSD), the standard deviation of all
RR intervals (SDNN), and the SDNN to RMSSD ratio
(SDNN/RMSSD) were calculated using a custom applica-
tion written in MATLAB (Version 8.4; MathWorks, Natick,
MA, USA) according to the formulas published by Wang
and Huang (2012). RMSSD is assumed to be an index of
vagal activity (Task Force of the European Society of Car-
diology, 1996), SDNN is assumed to be an index of total
variability (Aubert et al., 2003; Malik, 1997), and SDNN/
RMSSD is considered as an index of sympatho-vagal bal-
ance (Wang & Huang, 2012). Because raw HRV indexes
were not normally distributed, they were transformed using
natural logarithm (Ln).
Statistical analysis
MATLAB v8.4 with Statistics Toolbox v9.1 (MathWorks,
Natick, MA, USA) was used for statistical analyses. The
Kolmogorov-Smirnov test was used to verify that the stud-
ied variables have a normal distribution. The normal dis-
tribution was not rejected for all dependent variables (HR:
p = .23, Ln RMSSD: p = .36, Ln SDNN: p = .081, Ln
SDNN/RMSSD: p = .38) and therefore the use of statisti-
cal methods assuming a normal distribution was appropri-
ate. A linear model with one random factor (participants),
two fixed factors (beverage and time), and interaction (bev-
erage × time) was used to evaluate the effects of HRW and
time on the dependent variables. The beverage factor was
void in the baseline segment because neither HRW nor pla-
cebo was administered before the baseline segment. Fisher’s
LSD post-hoc test was used to calculate the significance of
the difference between the two selected means. For all tests,
p < .05 was considered statistically significant.
Arithmetic mean and standard deviation were used as
descriptive statistics. The partial eta-squared (ηp
2) was used
as an effect size for factors of the linear model and Cohen’s
d was used as an effect size for pairwise comparisons.
The magnitude of the effect size was interpreted accord-
ing following thresholds (Cohen, 1988): trivial (ηp
2 < .01,
d < 0.2), small (ηp
2 ≥ .01, d ≥ 0.2), medium (ηp
2 ≥ .06,
d ≥ 0.6), large (ηp
2 ≥ .14, d ≥ 1.2).
Results
A statistically significant beverage factor was found for HR
and Ln SDNN/RMSSD with medium and small effect
size, respectively (Table 2). A statistically significant time
Table 1 Chemical characteristic of hydrogen rich water and
placebo
Property HRW Placebo
pH 7.8 7.6
ORP (mV) –652 +170
Temperature (°C) 22 22
H2 concentration (ppm) 0.9 0.0
Note. HRW = hydrogen rich water; ORP = oxidation reduction potential.

4
M. Botek et al. Acta Gymnica, 2021, 51, e2021.009
factor was found for all dependent variables and the effect
size ranged from small to large (Table 2). The interaction
between beverage and time factor was not statistically sig-
nificant for any dependent variable (Table 2).
Pairwise comparisons are displayed in Figure 2. For
HR, pairwise comparisons showed significant differences
between HRW and placebo at R25 (HRW: 70.1 ± 11.5
beats · min–1, placebo: 66.9 ± 10.1 beats · min–1, p = .033,
d = 0.58, small effect) and R35 (HRW: 71.3 ± 9.9
beats · min–1, placebo: 67.9 ± 9.5 beats · min–1, p = .021,
d = 0.62, medium effect). HR at all recovery times after
both HRW and placebo administration was significantly
reduced compared to baseline (p ≤ .001, d ranging from
0.89 to 1.64, medium to large effects).
For Ln RMSSD, no statistically significant pair-
wise comparison between HRW and placebo was found
(p ≥ .20, d ranging from –0.35 to 0.11, trivial to small
effects). Ln RMSSD at all recovery times after both HRW
and placebo administration was significantly elevated com-
pared to baseline (p ≤ .004, d ranging from –1.57 to –0.80,
medium to large effects).
For Ln SDNN, a significant difference between HRW
and placebo was found in R45 (p = .049, d = 0.53, small
effect). Ln SDNN at all recovery times after HRW admin-
istration was significantly elevated compared to baseline
(p ≤ .001, d ranging from –1.16 to –0.90, large effects).
However, Ln SDNN after placebo administration was sig-
nificantly elevated only at R15, R25, and R35 (p ≤ .020, d
Table 2 Results of statistical analysis using a linear mixed-effects model
Variable Beverage factor Time factor Interaction
pηp
2pηp
2pηp
2
HR (beats · min–1) .002 .075 < .001 .403 .50 .020
Ln RMSSD (ms) .29 .009 < .001 .385 .64 .014
Ln SDNN (ms) .14 .019 < .001 .303 .28 .032
Ln SDNN/RMSSD .009 .057 .014 .100 .31 .030
Note. HR = heart rate; Ln = natural logarithm; RMSSD = square root of the mean of the squares of differences between adjacent RR intervals; SDNN = stan-
dard deviation of all RR intervals; SDNN/RMSSD = SDNN to RMSSD ratio.
Figure 2 Effect of hydrogen rich water (full circle) compared to placebo (empty circle) on heart rate (HR) and heart rate vari-
ability variables
Note. Values are presented as the mean and standard deviation. Ln = natural logarithm; RMSSD = square root of the mean of the squares of differ-
ences between adjacent RR intervals; SDNN = standard deviation of all RR intervals; SDNN/RMSSD = SDNN to RMSSD ratio; Bas = baseline segment
before any water administration; R15, R25, R35, and R45 = recovery segments 15, 25, 35, and 45 min respectively after drinking all the water.
*statistically significant (p < .05) difference between hydrogen rich water and placebo at the same time; †statistically significant (p < .05) difference between
this time and the baseline when hydrogen rich water was administered; ‡statistically significant (p < .05) difference between this time and the baseline when
placebo was administered.

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M. Botek et al. Acta Gymnica, 2021, 51, e2021.009
ranging from –1.32 to –0.63, large effects). The difference
at R45 was not statistically significant (p = .20, d = –0.34,
small effect).
For Ln SDNN/RMSSD, pairwise comparisons showed
significant differences between HRW and placebo at R25
(p = .049, d = 0.53, small effect) and R35 (p = .029,
d = 0.59, small effect). Ln SDNN/RMSSD after HRW
administration was significantly reduced at R15 (p = .012,
d = 0.69, medium effect) and returned at R25, R35, and
R45 to values not significantly different from baseline
(p ≥ .28, d ranging from 0.02 to 0.29, trivial to small
effects). However, Ln SDNN/RMSSD after placebo admin-
istration remained significantly reduced at all recovery times
(p ≤ .026, d ranging from 0.60 to 0.70, medium effects).
Discussion
The aim of this study was to assess the influence of acute
HRW intake on ANS activity response for the following
50 min in rest sitting. The main findings of the present
study revealed that 1260 ml of HRW compared to placebo
significantly modified autonomic cardiac regulation in
healthy females.
Specifically, in R15 of recovery after ingestion of 1260
ml HRW and/or placebo we observed a significant increase
in vagal activity with the concomitant reduction in HR
compared to baseline. A similar effect of acute water ingest
was reported also by Chen et al. (2004) whose results, taken
in a lying position revealed increased cardiac vagal activ-
ity after 500 ml of water administration. A significant rise
in HRV indicating a shift towards increased vagal activity
accompanied by reduced HR from 67.6 ± 2.0 to 60.7 ± 2.4
beats·min–1 was also found by Routledge et al. (2002) after
500 ml of water administration. Data in mentioned study
were measured in a supine position and reduction in HR
peaked between 20 and 25 min. In another study, Brown et
al. (2005) reported that 7.5 ml·kg–1 of water intake induced
an immediate increase in cardiac vagal activity and its
maintenance for at least 30 min as was found in our study.
The increase in vagal activity and concomitant decrease of
HR may be the underlying mechanism of reducing high
blood pressure which commonly occurs after water admin-
istration (Callegaro et al., 2007).
In contrast, in previous studies neither 480 ml (Jordan
et al., 2000) nor 500 ml (Schroeder et al., 2002) water
drinking in young healthy people did not cause changes
in blood pressure, and the authors concluded that water
drinking in healthy subjects may be an integrated response
consisting of increased sympathetic vasoconstrictor activ-
ity and a parallel increase in cardiac vagal activity. Thus,
the increase in peripheral resistance is counteracted by a
fall in cardiac output. Increased sympathetic activity after
water drinking confirmed Scott et al. (2001) who examined
muscle sympathetic neural activity, leading to peripheral
vasoconstriction. In our study, significantly reduced HR
compared to baseline value was found peaking at 35 min
after administration with a significant difference between
HRW and placebo. We assume that HR was reduced by the
influence of the same dose of water, however, the decrease
in HR after HRW administration was smaller compared to
placebo, likely due to the stimulating effect of HRW.
We found a significant shift in sympathovagal bal-
ance towards the sympathetic side 25 and 35 min after
HRW administration, whereas the vagal activity was
not significantly affected by HRW administration. We,
therefore, assume that the increase in HR was caused by
function changes in sympathetic activity. Interestingly, a
time-frame of the relative increase in sympathetic activity
after HRW ingestion that was detected in our study, notice-
ably matched with dynamics of the H2 peak in the breath
in humans (Shimouchi et al., 2009). We explain the loss
of significant differences in HR and Ln SDNN/RMSSD
beyond 35 min by the utilizing of H2 in the human body
or by exhaling of H2 from the body. The stimulating effect
of HRW on brain circuits was recently found also by Zanini
et al. (2020) who examined markers of alertness in sleep
deprived men and women. Mentioned authors reported a
similar effect of HRW compared to caffeine when HRW
positively affected orienting to sensory stimulation and con-
cluded that HRW might be advanced as a safe and effective
alternative to caffeine. In contrast to our results, Mizuno et
al. (2017) reported an attenuation in sympathetic activity
(low frequency) during the resting state in adult volunteers
after 4-week administration of 600 ml HRW per day and
concluded that besides autonomic function, HRW intake
may increase central nervous system functions improving
mood and anxiety. In regard to ANS activity, discrepancies
between our results and the study mentioned above might
be explained by a different period of HRW administra-
tion. From a practical point of view, we feel that if acute
H2 administration may stimulate the sympathetic cardiac
system, therefore it would not be recommended to ingest
HRW at least 60 min before sleeping in order to avoid
issues with falling asleep.
One limitation of this study might be the lack of infor-
mation about blood pressure response to pre and post HRW
administration. Another limit is that we used a constant
amount of HRW per subject and we did not adjust it to the
body mass of subjects. The menstrual cycle was not evalu-
ated in this study, which can be considered as another limit
as well as the low sample size.
Conclusions
Acute ingestion of 1260 ml of HRW in healthy females
induced a relative increase in sympathetic cardiac activity
between 25 and 35 min post-ingestion, whereas vagal activ-
ity was not affected by H2 administration.
Acknowledgments
This study was supported by Palacký University Olomouc
(grant number IGA_FTK_2020_007). The authors would
like to thank Hana Baleková for collecting data for this study.
Conflict of interest
The authors report no conflict of interest.

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M. Botek et al. Acta Gymnica, 2021, 51, e2021.009
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