Hydrogen-rich water for improvements of mood, anxiety, and autonomic nerve function in daily life

Abstract

Health and a vibrant life are sought by everyone. To improve quality of life (QOL), maintain a healthy state, and prevent various diseases, evaluations of the effects of potentially QOL-increasing factors are important. Chronic oxidative stress and inflammation cause deteriorations in central nervous system function, leading to low QOL. In healthy individuals, aging, job stress, and cognitive load over several hours also induce increases in oxidative stress, suggesting that preventing the accumulation of oxidative stress caused by daily stress and daily work contributes to maintaining QOL and ameliorating the effects of aging. Hydrogen has anti-oxidant activity and can prevent inflammation, and may thus contribute to improve QOL. The present study aimed to investigate the effects of drinking hydrogen-rich water (HRW) on the QOL of adult volunteers using psychophysiological tests, including questionnaires and tests of autonomic nerve function and cognitive function. In this double-blinded, placebo-controlled study with a two-way crossover design, 26 volunteers (13 females, 13 males; mean age, 34.4 ± 9.9 years) were randomized to either a group administered oral HRW (600 mL/d) or placebo water (PLW, 600 mL/d) for 4 weeks. Change ratios (post-treatment/pre-treatment) for K6 score and sympathetic nerve activity during the resting state were significantly lower after HRW administration than after PLW administration. These results suggest that HRW may reinforce QOL through effects that increase central nervous system functions involving mood, anxiety, and autonomic nerve function.

Full text

© 2017 Medical Gas Research | Published by Wolters Kluwer - Medknow 247
RESEARCH ARTICLE
Hydrogen-rich water for improvements of mood, anxiety, and
autonomic nerve function in daily life
Kei Mizuno1-5, Akihiro T. Sasaki1-3, 6, Kyoko Ebisu1, 3, Kanako Tajima2, 3, Osami Kajimoto1, 5, Junzo Nojima7,
Hirohiko Kuratsune8, Hiroshi Hori1-3, Yasuyoshi Watanabe1-4, 6, *
1 Osaka City University Center for Health Science Innovation, Osaka, Japan
2 Pathophysiological and Health Science Team, RIKEN Center for Life Science Technologies, Kobe, Japan
3 Health Evaluation Team, RIKEN Compass to Healthy Life Research Complex Program, Kobe, Japan
4 Health Metrics Development Team, RIKEN Compass to Healthy Life Research Complex Program, Kobe, Japan
5 Department of Medical Science on Fatigue, Osaka City University Graduate School of Medicine, Osaka, Japan
6 Department of Physiology, Osaka City University Graduate School of Medicine, Osaka, Japan
7
Department of Laboratory Science, Faculty of Health Science, Yamaguchi University Graduate School of Medicine, Yamaguchi, Japan
8 Department of Health Science, Faculty of Health Science for Welfare, Kansai University of Welfare Sciences, Kashihara, Japan
*Correspondence to: Yasuyoshi Watanabe, M.D., Ph.D., yywata@riken.jp.
Health and a vibrant life are sought by everyone. To improve quality of life (QOL), maintain a healthy state, and prevent various diseases,
evaluations of the effects of potentially QOL-increasing factors are important. Chronic oxidative stress and inammation cause deteriora-
tions in central nervous system function, leading to low QOL. In healthy individuals, aging, job stress, and cognitive load over several
hours also induce increases in oxidative stress, suggesting that preventing the accumulation of oxidative stress caused by daily stress and
daily work contributes to maintaining QOL and ameliorating the effects of aging. Hydrogen has anti-oxidant activity and can prevent
inammation, and may thus contribute to improve QOL. The present study aimed to investigate the effects of drinking hydrogen-rich
water (HRW) on the QOL of adult volunteers using psychophysiological tests, including questionnaires and tests of autonomic nerve
function and cognitive function. In this double-blinded, placebo-controlled study with a two-way crossover design, 26 volunteers (13
females, 13 males; mean age, 34.4 ± 9.9 years) were randomized to either a group administered oral HRW (600 mL/d)
or placebo water
(PLW, 600 mL/d) for 4 weeks. Change ratios (post-treatment/pre-treatment) for K6 score and sympathetic nerve activity during the
resting state were signicantly lower after HRW administration than after PLW administration. These results suggest that HRW may
reinforce QOL through effects that increase central nervous system functions involving mood, anxiety, and autonomic nerve function.
Key words: anxiety; autonomic nerve function; hydrogen-rich water; mood; quality of life
doi: 10.4103/2045-9912.222448
How to cite this article: Mizuno K, Sasaki AT, Ebisu K, Tajima K, Kajimoto O, Nojima J, Kuratsune H, Hori H, Watanabe Y.
Hydrogen-rich water for improvements of mood, anxiety, and autonomic nerve function in daily life. Med Gas Res. 2017;7(4):247-255.
Abstract
IntroductIon
Health and a vibrant life are much craved by everyone. To
improve quality of life (QOL), maintain a healthy state,
and prevent the onset of various diseases, evaluation of
interventional effects for improving QOL is important. The
high metabolic rate of the brain results in the generation of
disproportionate amounts of reactive oxygen and nitrogen
species, leading to increased oxidative stress.1 Increased
oxidative stress and lipid peroxidation initiate a cascade of
proinammatory signals, leading to inammation. Altered
homeostasis of oxidation, inammation, and protein ag-
gregation has been suggested to contribute to the death of
neurons, which is directly related to impairments in various
cognitive domains. As such, chronic oxidative stress and
inammation may cause deteriorations in the function of
the central nervous system, leading to reductions in QOL.
Hydrogen has antioxidant activity and can prevent inam-
mation.2-4 The distribution of hydrogen throughout the

Medical Gas Research ¦ December ¦ Volume 7 ¦ Issue 4
248
Mizuno et al. / Med Gas Res www.medgasres.com
brain
and body indicates actions both in the central and
peripheral nervous systems. Previous clinical studies
have shown that hydrogen-rich water (HRW) reduces
concentrations of markers of oxidative stress in patients
with metabolic syndrome,
5,6
improves lipid and glucose
metabolism in patients with type 2 diabetes,
7
improves
mitochondrial dysfunction in patients with mitochon-
drial myopathies, and reduces inammatory processes
in patients with polymyositis/dermatomyositis.
8
In an-
other study, exercise-induced declines in muscle function
among elite athletes were also improved by administering
HRW.
9
Although such ndings suggest that HRW may
help alleviate symptoms of several diseases and increase
the physical performance of athletes, the effects of pro-
longed HRW ingestion on the QOL of individuals in the
general population remain unknown.
Some reports have demonstrated that oxidative stress
is associated with QOL in patients with chronic obstruc-
tive pulmonary disease and cervical cancer.
10,11
During
oncological treatment among patients with cervical
cancer, antioxidant supplementation was found to be
effective in improving QOL.
11
In addition, Kang et al.
12
reported that treatment with HRW for patients receiving
radiotherapy for liver tumors decreased oxidative stress
and improved QOL. Although the association between
oxidative stress and QOL in healthy individuals is still
unclear, aging, job stress, and cognitive load over the
course of several hours in healthy individuals have also
been found to induce increases in oxidative stress,
13-16
suggesting that preventing the accumulation of oxida-
tive stress caused by daily stress and daily work may
contribute to the maintenance of QOL and amelioration
of the effects of aging. Continuous HRW intake might
therefore be expected to reduce accumulation of oxida-
tive stress, thus helping to prevent decreases in QOL.
The aim of the present study was to investigate the effects
of drinking 600 mL of HRW per day for 4 weeks on the
QOL of adult volunteers using questionnaires for sleep,
fatigue, mood, anxiety, and depression, an autonomic
function test, and a higher cognitive function test.
subjects and Methods
Subjects
Thirty-one adult volunteers between 20 and 49 years old
participated in this double-blinded, randomized, placebo-
controlled study with a two-way crossover design. Exclu-
sion criteria comprised: history of chronic illness; chronic
medication or use of supplemental vitamins; employment
in shift work; pregnancy; body mass index ≤ 17 or ≥ 29
kg/m2; food allergy; history of smoking; or history of
drinking excessive amounts of alcohol (≥ 60 g/day). Shift
workers were excluded because the water was administered
at breakfast and dinner, the timings of which are irregular
among shift workers. In addition, the mental and physical
conditions of shift workers can be greatly affected by the
shift schedule for the preceding 2 days, which may impact
the results obtained from the questionnaires used in this
study. Before each experiment, participants were asked
to refrain from drinking alcohol, since drinking excessive
amounts of alcohol carries signicant risks of uctuations
in physical condition. All experiments were conducted in
compliance with national legislation and the Code of Ethical
Principles for Medical Research Involving Human Subjects
of the World Medical Association (the Declaration of Hel-
sinki) and registered to the UMIN Clinical Trials Registry
(No. UMIN000022382). The study protocol was approved
by the Ethics Committee of Osaka City University Center
for Health Science Innovation (OCU-CHSI-IRB No. 4),
and all participants provided written informed consent for
participation in the study.
Study design
We used a double-blinded, placebo-controlled study with
a two-way crossover design, as summarized in Figure 1.
After admission to the study, participants were randomized
in a double-blinded manner to receive HRW in an aluminum
pouch (0.8–1.2 ppm of hydrogen, 300 mL/pouch; Melodian
Corporation, Yao, Japan) or placebo water (PLW), repre-
senting mineral water from the same source (i.e., same
components without hydrogen) in an aluminum pouch
(0 ppm of hydrogen, 300 mL/pouch; Melodian Corpora-
Figure 1: Time course of the experiments.
Note: Participants were randomly divided into two study groups.
The experiment consisted of 4 weeks of hydrogen-rich water
(HRW) administration or placebo water (PLW) administration,
a 4-week washout period, and then another 4 weeks of PLW
administration or HRW administration. Before (pre) and after
(post) each period of HRW or PLW administration, subjective
and objective measurements for quality of life were obtained,
such as results for sleep, mood, anxiety, feelings of depression,
autonomic nerve function, and cognitive function.

Medical Gas Research ¦ December ¦ Volume 7 ¦ Issue 4 249
Mizuno et al. / Med Gas Res www.medgasres.com
Studies Depression Scale.21 General sleepiness and daytime
sleepiness scores were calculated using the Pittsburgh Sleep
Quality Index (PSQI)22 and the Epworth Sleepiness Scale,23
respectively. The reliability and validity of the Japanese
versions of these questionnaires have been conrmed.19,24-28
Autonomic function test
Participants underwent simultaneous electrocardiography
and photoplethysmography using a Vital Monitor 302
system (Fatigue Science Laboratory, Osaka, Japan) while
sitting quietly with their eyes closed for 3 minutes. These
data were analyzed using MemCalc software (GMS, Tokyo,
Japan). Frequency analyses for R-R interval variation from
electrocardiography and a-a interval variation as the second
derivative of photoplethysmography (accelerated plethys-
mography) were performed using the maximum entropy
method, which is capable of estimating the power spectrum
density from short time series data, and is adequate for
examining changes in heart rate variability under different
conditions of short duration.29,30 The power spectrum resolu-
tion was 600 Hz. For frequency analyses, the low-frequency
component power (LF) was calculated as the power within
a frequency range of 0.04–0.15 Hz, and the high-frequency
component power (HF) was calculated as that within a fre-
quency range of 0.15–0.4 Hz. HF is vagally mediated,31-33
whereas LF originates from a variety of sympathetic and
vagal mechanisms.30,34 Some review articles35-37 mentioned
that LF reects sympathetic nerve activity and is used as
a marker of sympathetic nerve activity in original articles.
Before autonomic nerve function testing was conducted
for 3 minutes, a practice test was conducted for a period
of 1 minute, in accordance with previous studies.38-40 The
reliability of these tests has been conrmed.41,42
Cognitive function test
Since previous studies have revealed that a switching at-
tention task is useful for evaluating reduced performance
under fatigue conditions,43-45 we used task E of the modi-
ed advanced trail making test (mATMT) as a switching
attention task for evaluating executive function.46,47 Circles
with numbers (from 1 to 13) or kana (Japanese phonograms,
12 different letters) were shown in random locations on a
screen, and participants were required to use a computer
mouse to alternately touch the numbers and kana; this
task thus required switching attention. When participants
touched a target circle, it remained in the same position,
but its color changed from black to yellow. Participants
were instructed to perform the task as quickly and correctly
as possible, and continuously performed this task for 5
minutes. We evaluated three indices of task performance:
the total count of correct responses (number of correctly
tion) twice a day for 4 weeks. Fifteen participants were
administered PLW rst, and then HRW. The remaining 16
participants were administered HRW rst, and then PLW.
Participants consumed water within 5 minutes twice a day,
at breakfast and dinner in their home, and conrmed the
water intake at breakfast and dinner in a daily journal for
4 weeks. We assessed the intake rate of water by checking
the daily journal every 4 weeks, on the 2nd and 4th experi-
mental days. No participants reported any difference in taste
between HRW and PLW. Previous studies have reported
interventional effects of administering HRW to humans at
hydrogen concentrations under 1.3 ppm.5,12 We therefore
used a similar concentration of 0.8–1.2 ppm in the present
study. Absolute volumes (600 mL) of HRW and PLW were
provided to participants rather than a volume proportional
to body mass, based on previously reported results.5-7,12
The duration of supplementation was set based on previous
ndings with HRW administration for 2–8 weeks.5,12,17 A
4-week washout period was provided between HRW and
PLW administrations based on a previous study.8 The day
before starting each experiment, participants were told to
nish dinner by 21:00, and were required to fast overnight
to avoid any inuence of diet on concentrations of mea-
sured parameters (markers of inammation and oxidative
stress) in blood samples. At 09:00 the next day, participants
completed the questionnaires after conrming that they had
refrained from drinking alcohol, had nished dinner by
21:00, and had fasted overnight. Autonomic nerve func-
tion was measured at 09:30. Cognitive function testing was
conducted at 09:45. Blood samples were collected at 10:00.
These measurements were performed a total of four times
for each participant, before (pre) and after (post) each of
the two 4-week administration periods. From 24 hours (the
day before the visit day) before each visit for measurements,
participants were told to refrain from drinking alcohol or
performing strenuous physical activity and to follow their
normal diets, drinking habits, and sleeping hours. During the
4-week PLW or HRW administration periods, daily daytime
activity (amount of physical exertion) of participants was
measured using a pedometer and participants kept a daily
journal to record drinking volume and times of PLW or
HRW intake, physical condition (e.g., pain, lassitude, and
indenite complaints), sleeping times, etc.
Questionnaire
Severity of fatigue was measured using the Chalder Fatigue
Scale (CFS)18 and a modied version of the Osaka City
University Hospital Fatigue Scale.19 Mood and anxiety
were evaluated using the K6 scale.20 Symptoms of depres-
sion were measured using the Center for Epidemiologic

Medical Gas Research ¦ December ¦ Volume 7 ¦ Issue 4
250
Mizuno et al. / Med Gas Res www.medgasres.com
distributions) of each measured parameter using the Kol-
mogorov-Smirnov test. Values are presented as the mean
± standard deviation or median and interquartile range
based on the results of Kolmogorov-Smirnov test. The
Wilcoxon signed-rank test for non-parametric parameters
and paired t-test for differences between HRW and PLW
administrations after two-way repeated-measurement
analysis of variance for parametric parameters were con-
ducted. If signicant changes were observed by compari-
sons within each condition (pre- vs. post-HRW; pre- vs.
post-PLW) or between post-treatment values (post-HRW
vs. post-PLW), then we compared change ratios between
post-HRW/pre-HRW and post-PLW/pre-PLW using the
Wilcoxon signed-rank test or paired t-test. All P values
were two-tailed, and those less than 0.05 were considered
statistically signicant. Statistical analyses were performed
using IBM SPSS Statistical Package version 20.0 (IBM,
Armonk, NY, USA).
results
General results
During the study, we excluded ve participants from data
analyses due to symptoms of hay fever, prolonged medica-
tion use because of a cold, insufcient intake of HRW or
PLW intake (≥ 85%), or a frequency of special events ≤ 15%
as recorded in the daily diary. We thus analyzed data from
a total of 26 participants (13 females, 13 males; mean age,
34.4 ± 9.9 years; mean body mass index, 21.5 ± 2.6 kg/m
2
).
No side, order, and carry-over effects were observed from
the oral administrations of HRW and PLW in any participant.
Questionnaire results
Results from the questionnaires are summarized in Table 1.
No
questionnaire scores at baseline (pre) showed any signicant
differences between HRW and PLW administration groups.
With HRW administration, scores for K6, CFS, and PSQI
were signicantly decreased after the 4-week administration
period. In addition, the change ratio (post/pre) for K6 score
was signicantly lower in the HRW administration group
than in the PLW administration group (Figure 2). No sig-
nicant changes were seen in any other questionnaire scores
(modied version of the Osaka City University Hospital
Fatigue Scale, Center for Epidemiologic Studies Depression
Scale or Epworth Sleepiness Scale) after HRW administration
and no signicant changes in any of the scores were seen after
PLW administration. Likewise, these scores did not differ
signicantly between HRW and PLW after administration.
Autonomic function results
Results for the autonomic nerve function are summarized
in Table 1. LF, HF, and LF/HF ratio at baseline (pre) did
touched numbers and letters); the total count of errors
(number of incorrectly touched numbers and letters); and
the motivational response (reaction time from a nished
trial to the next trial). Based on our previous study,47 be-
fore participants performed task E of the mATMT on each
experimental day, they practiced for a period of 1 minute.
The reliability of this test has been conrmed.43,44
Blood sample analyses
Blood samples were collected from the brachial vein. The
amount of blood sampled was 13 mL per experimental day.
We thus collected blood samples on four occasions (once
per experimental day) in the study. Blood samples for serum
analyses were centrifuged at 1,470 × g for 5 minutes at 4°C.
The concentration of high-sensitivity C-reactive protein
(hs-CRP) in each serum sample was assessed by particle-
enhanced immunonephelometry using a BNII analyzer (BN
II ProSpec; Siemens, Munich, Germany). Oxidative activity
in each serum sample was assessed with the reactive oxygen
metabolites-derived compounds (d-ROMs) test (Diacron
International, Grosseto, Italy), while anti-oxidative activ-
ity was measured with the biological anti-oxidant potential
(BAP) test (Diacron International) using a JCABM1650
automated analyzer (JEOL, Tokyo, Japan).48 The concentra-
tions of ROMs are expressed in Carratelli units (1 CARR U
= 0.08 mg of hydrogen peroxide/dL).49 The oxidative stress
index (OSI) was calculated using the following formula:
OSI = C × (d-ROMs/BAP), where C denotes a coefcient
for standardization to set the mean OSI in healthy individu-
als at 1.0 (C = 8.85).45 All supernatants were stored at -80°C
until analyzed. Assays for hs-CRP were performed at LSI
Medience Corporation (Tokyo, Japan) and those for serum
d-ROMs and BAP were performed at Yamaguchi University
Graduate School of Medicine.
Daily daytime activity and daily journal
Daily daytime activity, representing the expenditure of
calories and amount of physical activity (METs × time) was
recorded using an Active style Pro HJA-350IT pedometer
(OMRON, Kyoto, Japan). A daily journal was kept for 4
weeks, and included information on fatigue (based on a
visual analogue scale from 0, representing “no fatigue”,
to 100, representing “total exhaustion”) just after waking
up and before bedtime, sleeping times, physical condition
(1, good; 2, normal; or 3, bad), and special events (if the
day was different from a usual day: 1, no; or 2, yes). We
carefully checked the daily journal every four weeks, on
the 2nd, 3rd, and 4th experimental days.
Statistical analyses
First, we tested the normality (parametric or non-parametric

Medical Gas Research ¦ December ¦ Volume 7 ¦ Issue 4 251
Mizuno et al. / Med Gas Res www.medgasres.com
not differ significantly between HRW and PLW admin-
istrations, indicating similar autonomic nerve function
in the two groups before water intake. Although the
HF and LF/HF ratio were not significantly affected by
4-week administrations of HRW or PLW, LF after HRW
administration was significantly lower than that after
PLW administration. The change ratio (post/pre) for LF
was also significantly lower in the HRW administration
group than in the PLW administration group (Figure 2).
Cognitive function results
Results for the cognitive function test are shown in
Table 1. Motivational response and total counts of cor-
rect responses and errors at baseline (pre) did not differ
signicantly between HRW and PLW administrations,
indicating similar cognitive function between groups
Table 1: Changes in parameters related to quality of life due to hydrogen-rich water (HRW) or placebo water (PLW)
administration
HRW P LW
Pre Post Pre Post
Questionnaire
CFS 2.0 (0–4.8) 0 (0–3.8)* 2.5 (0–5.8) 0.5 (0–4.0)
mOCUH-FS 70.6±21.5 68.2±18.3 68.2±21.4 67.9±22.5
K6 8.5 (6.0–12.8) 7.0 (6.0–10.0)* 7.0 (6.0–12.3) 8.5 (6.0–11.0)
CES-D 6.0 (4.3–13.8) 9.0 (4.0–16.0) 8.5 (4.0–16.8) 9.0 (4.0–16.0)
PSQI 4.0 (3.3–6.8) 3.0 (3.0–4.8)* 3.5 (3.0–5.8) 4.0 (3.0–5.0)
ESS 9.3±4.9 9.6±5.6 9.7±4.9 9.0±5.1
Autonomic function
LF (ms2)519 (268–1,152) 426 (290–594)† 377 (270–639) 504 (242–1,004)
HF (ms2)239 (185–559) 219 (124–403) 249 (97–519) 243 (170–345)
LF/HF 1.9 (1.1–4.5) 2.0 (1.0–3.6) 1.8 (0.9–3.6) 1.9 (1.3–3.0)
Task E of mATMT
TCCR (number) 167.5±42.2 175.2±38.8 166.3±35.6 175.5± 8.5
TCE (number) 1.0 (0.5–2.2) 0.9 (0.5–1.6) 1.4 (0.7–2.3) 1.6 (0.6–2.2)
MR (second) 0.83 (0.67–0.93) 0.73 (0.62–0.86)* 0.75 (0.69–0.92) 0.78 (0.65–0.91)
Biochemical marker
hs-CRP (mg/L) 0.02 (0.01–0.04) 0.02 (0.01–0.04) 0.02 (0.01–0.04) 0.01 (0.01–0.03)
d-ROMs (CARR U) 301.1±47.3 297.3±48.8 300.3±53.1 303.9±51.2
BAP (µM) 2,645±231 2,598±252 2,683±232 2,660±274
OSI 1.02±0.19 1.02±0.20 1.00±0.20 1.02±0.21
Note: CFS: Chalder Fatigue Scale; mOCUH-FS: modified version of the Osaka City University Hospital Fatigue Scale; CES-D: The Center for
Epidemiologic Studies Depression Scale; PSQI: the Pittsburgh Sleep Quality Index; ESS: Epworth Sleepiness Scale; HF: high-frequency component
power; LF/HF: the ratio of low-frequency component power and HF; mATMT: modified advanced trail making test; TCCR: total counts of correct
responses; TCE: total counts of errors; MR: Motivational response; hs-CRP: high-sensitivity C-reactive protein; d-ROMs: reactive oxygen metabolites-
derived compounds; CARR U: Carratelli units ; BAP: biological anti-oxidant potential; OSI: oxidative stress index; Pre: before the 4-week administration
period; Post: after the 4-week administration period. Values are shown as the mean ± standard deviation or medians (inter-quartile ranges). *P < 0.05,
vs. Pre conditions; †P < 0.05, vs. PLW.
Figure 2: Comparison of
change ratios (post-treatment/
pre-treatment) for parameters
related to quality of life with
administration of hydrogen-rich
water (HRW) or placebo water
(PLW) for 4 weeks.
Note: Change ratios for K6 score
for mood (A) and anxiety and the
low-frequency component power
(LF) for autonomic nerve function
(B). *P < 0.05.
AB
–

Medical Gas Research ¦ December ¦ Volume 7 ¦ Issue 4
252
Mizuno et al. / Med Gas Res www.medgasres.com
before water intake. Motivational response after HRW
administration was signicantly faster than that before
HRW administration. The change ratio (post/pre) for
motivational response was not signicantly different in
the HRW administration group than in the PLW adminis-
tration group. No signicant differences in motivational
response, total counts of correct responses, or errors
after water administration were seen between HRW- and
PLW-administered conditions.
Blood sample results
No signicant differences were seen in any blood param-
eters (hs-CRP, d-ROMs, BAP, and OSI) before HRW or
PLW administration (Table 1), indicating the comparability
of the two groups before water intake. After HRW and PLW
administrations, we again found no signicant differences
in these blood parameters.
Daily daytime activity and daily journal results
The daily expenditure of calories and amount of physical
activity during the 4-week administration periods did not
differ signicantly between HRW and PLW administration
conditions (Table 2). Similarly, visual analogue scale scores
for fatigue just after waking and before bedtime, sleeping
times, physical condition, and counts of special events
were comparable between HRW and PLW administration
conditions (Table 2), indicating that living habits were
successfully controlled during the experimental period in
the two groups.
Table 2: Daily daytime activity and data recorded in the
daily journal during the hydrogen-rich water (HRW) or
placebo water (PLW) administration period (4 weeks)
HRW P LW
Daily daytime activity
Expenditure of calories (kcal)
2,071±306 2,082±299
Physical activity
(
METs ×
time)
4.07±1.66 4.27±1.59
Daily journal
VAS-F of wake-up 38.7±20.4 35.9±17.8
VAS-F of bedtime 35.7±21.7 33.4±17.5
Physical condition
2.00 (2.00–2.04) 2.00 (2.00–2.04)
Special event
1.04 (1.00–1.11) 1.00 (1.00–1.10)
Note: METs: Metabolic equivalents; VAS-F: visual analogue scale for
fatigue. Values are shown as the mean ± standard deviation or medians
(inter-quartile ranges).
dIscussIon
The present ndings suggest that HRW administration for 4
weeks may have improved the QOL of adult volunteers in
terms of improved mood and anxiety and reduced activity
of the sympathetic nervous system at rest.
In terms of associations between hydrogen and the central
nervous system, a report by Ohsawa et al.
4
was the rst to
demonstrate that molecular hydrogen acts, at least in part, as
an anti-oxidant as it binds to hydroxyl ions produced in central
nervous system injuries. Previous studies have proposed that
HRW administration has neuroprotective effects
50
and anti-
aging effects on periodontal oxidative damage in healthy aged
rats.
51
In a rat model of Alzheimer’s disease, hydrogen-rich
saline prevented neuroinammation and oxidative stress,
and improved memory function.
52
In terms of the association
between HRW and QOL, only one study reported that HRW
administration for 6 weeks improved QOL scores in patients
treated with radiotherapy for liver tumors.
12
Although reports
on the effects of HRW administration in healthy populations
have not been accumulated, job stress
14,15
and acute fatigue
caused by mental and physical loading for several hours
16,53
have been shown to enhance oxidative stress. As for physical
fatigue, in order to alleviate acute physical fatigue in healthy
volunteers not including athletes, we have previously demon-
strated that treatment with antioxidant supplements is effec-
tive.
54-56
The present study provided new ndings that HRW
affects not only physical condition but also mental conditions
such as mood, anxiety, and autonomic nerve function. One of
the advantages of HRW is the ability to cross the blood-brain
barrier, offering high potential to reduce oxidative stress in the
brain. A previous study in rats found that levels of malondi-
aldehyde, a marker of oxidative stress, were around 4.8-fold
higher in the brain than in the blood (plasma).
57
These results
suggest that HRW may be effective for reducing accumulated
oxidative stress in the brain in daily life, potentially contribut-
ing to the maintenance of central nervous system activity and
preventing decreases in QOL.
In the present study, mood and anxiety levels improved
after HRW administration. These negative emotions are
also known to be involved in conditions related to oxidative
stress; social phobia,58,59 depression,60 anxiety,61,62 and other
neuropsychiatric disorders63 have been shown to be associ-
ated with increased oxidative stress. Neuroinammation
is also related to fatigue, mood, anxiety, and sleep.64-67 In
older mice, HRW administration succeeded in suppressing
depression-like behaviors.68 These ndings suggest that ad-
ministration of HRW for 4 weeks may be effective for con-
trolling such negative emotions by reducing oxidative stress
and inammation of the central nervous system. Increasing
evidence suggests that oxidative stress and inammation
in neurons are involved in the pathological manifestations
of many neurological and neuropsychiatric disorders, and
HRW administration may thus help alleviate the symptoms
of these disorders. Previous study revealed that oxidative
stress of the brain causes cognitive and motivational decits
in a mouse model of neuropsychiatric disorder (schizo-

Medical Gas Research ¦ December ¦ Volume 7 ¦ Issue 4 253
Mizuno et al. / Med Gas Res www.medgasres.com
phrenia).69 In the present study, motivational response of
cognitive function test was improved by prolonged HRW
intake, suggesting that a reduction of oxidative stress in
the brain by the intake of HRW may increase motivational
performance of cognitive task.
Stressors can enhance sympathetic hyperactivity, promote
oxidative stress, and boost pro-inammatory cytokine pro-
duction.
70-72
Autonomic nerve function is thus closely associ-
ated with oxidative stress and inammation. Attenuation of
sympathetic nervous system activity during the resting state
in adult volunteers may therefore be the result of decreases
in inammation and oxidative stress as an effect of pro-
longed HRW administration. However, the lack of changes
in oxidative stress markers noted in the present study after
HRW intake for 4 weeks could be due to the low severity of
oxidative stress in the participants. Actually, serum d-ROMs
(307.1 ± 49.4 CARR U) and BAP (2,549 ± 194 µM) concen-
trations at the rst measurement point in the present study
were within normal ranges based on the results of serum d-
ROMs (286.9 ± 100.2 CARR U) and BAP (2,541 ± 122 µM)
concentrations measured in 312 healthy participants in our
previous study.
48
However, levels of oxidative stress uctuate
depending on daily work load and stress. In addition, the rat
study by García-Niño et al.
57
that found malondialdehyde
levels around 4.8-fold higher in the brain than in plasma
indicate that oxidative stress in the brain is more severe.
Daily administration of HRW for 4 weeks may thus con-
tribute to attenuation of and prevention from the cumulative
oxidative stress in the brain. Mood, anxiety, and autonomic
nerve function could thus potentially be improved. Although
the range of sympathetic nerve activity in the present study
considers to be normal based on our previous studies,
73,74
sympathetic nerve activity also uctuates depending on
daily work load and stress.
35
Therefore, lower sympathetic
nerve activity of resting state may contribute to suppress an
excessive increase in sympathetic nerve activity after the
daily work load and stress.
We conducted this study with a limited number of par-
ticipants. Before our results can be generalized, studies
involving larger numbers of participants are essential.
Although we mainly examined the effects of HRW on
the central nervous system, we did not directly evaluate
the dynamics of inammation and oxidation in the brain.
Neuroimaging studies using positron emission tomography
and magnetic resonance imaging are thus underway in
our laboratory to identify the mechanisms underlying the
effects of HRW intake on the central nervous system that
can improve QOL.
In conclusion, HRW administration for 4 weeks in adult
volunteers improved mood, anxiety, and autonomic nerve
function, suggesting that HRW administration may offer
an effective method to reinforce QOL and maintain good
health. In a further study, we will try to identify the effects
of HRW administration in participants with ongoing stress
or chronic fatigue.
Acknowledgments
We would like to thank Ms. Mika Furusawa for her excellent tech-
nical assistances and Forte Science Communications for editorial
help with this manuscript.
Author contributions
KM, OK, HK, and YW conceived and designed the experiments;
KM, ATS, KE, and HH performed the experiments; KM, ATS, KE,
KT, and JN analyzed the data; and KM, ATS, and YW wrote the
paper. All the authors approved the nal version of manuscript.
Conflicts of interest
This work was presented at Japanese Society of Fatigue Science,
Yamaguchi City, Japan on May 16, 2016. Yasuyoshi Watanabe
received funding for the present study from Melodian Corporation.
The other authors have no conicts of interest to declare.
Research ethics
All experiments were conducted in compliance with national leg-
islation and the Code of Ethical Principles for Medical Research
Involving Human Subjects of the World Medical Association (the
Declaration of Helsinki) and registered to the UMIN Clinical Trials
Registry (UMIN000022382). The study protocol was approved by
the Ethics Committee of Osaka City University Center for Health
Science Innovation (OCU-CHSI-IRB No. 4).
Declaration of participant consent
The authors certify that they have obtained all appropriate partici-
pant consent forms. In the form the participants have given their
consent for their images and other clinical information to be re-
ported in the journal. The participants understand that their names
and initials will not be published and due efforts will be made to
conceal their identity, but anonymity cannot be guaranteed.
Data sharing statement
Datasets analyzed during the current study are available from the
corresponding author on reasonable request.
Plagiarism check
Checked twice by iThenticate.
Peer review
Externally peer reviewed.
Open access statement
This is an open access article distributed under the terms of the
Creative Commons Attribution-NonCommercial-ShareAlike 3.0
License, which allows others to remix, tweak, and build upon the
work non-commercially, as long as the author is credited and the
new creations are licensed under identical terms.
Open peer reviewers
Lei Huang, Loma Linda University, USA; Qin Hu, Shanghai Jiao
Tong University, China.
references
1. Starke RM, Chalouhi N, Ali MS, et al. The role of oxidative
stress in cerebral aneurysm formation and rupture. Curr Neuro-
vasc Res. 2013;10:247-255.

Medical Gas Research ¦ December ¦ Volume 7 ¦ Issue 4
254
Mizuno et al. / Med Gas Res www.medgasres.com
2.
Buchholz BM, Kaczorowski DJ, Sugimoto R, et al. Hydrogen
inhalation ameliorates oxidative stress in transplantation induced
intestinal graft injury. Am J Transplant. 2008;8:2015-2024.
3. Huang CS, Kawamura T, Toyoda Y, Nakao A. Recent advances
in hydrogen research as a therapeutic medical gas. Free Radic
Res. 2010;44:971-982.
4. Ohsawa I, Ishikawa M, Takahashi K, et al. Hydrogen acts as a
therapeutic antioxidant by selectively reducing cytotoxic oxy-
gen radicals. Nat Med. 2007;13:688-694.
5. Nakao A, Toyoda Y, Sharma P, Evans M, Guthrie N. Effective-
ness of hydrogen rich water on antioxidant status of subjects
with potential metabolic syndrome-an open label pilot study. J
Clin Biochem Nutr. 2010;46:140-149.
6.
Song G, Li M, Sang H, et al. Hydrogen-rich water decreases serum
LDL-cholesterol levels and improves HDL function in patients with
potential metabolic syndrome. J Lipid Res. 2013;54:1884-1893.
7. Kajiyama S, Hasegawa G, Asano M, et al. Supplementation of
hydrogen-rich water improves lipid and glucose metabolism
in patients with type 2 diabetes or impaired glucose tolerance.
Nutr Res. 2008;28:137-143.
8. Ito M, Ibi T, Sahashi K, Ichihara M, Ito M, Ohno K. Open-
label trial and randomized, double-blind, placebo-controlled,
crossover trial of hydrogen-enriched water for mitochondrial
and inammatory myopathies. Med Gas Res. 2011;1:24.
9.
Aoki K, Nakao A, Adachi T, Matsui Y, Miyakawa S. Pilot study:
Effects of drinking hydrogen-rich water on muscle fatigue caused
by acute exercise in elite athletes. Med Gas Res. 2012;2:12.
10. Ben Moussa S, Rouatbi S, Ben Saad H. Incapacity, handicap,
and oxidative stress markers of male smokers with and without
COPD. Respir Care. 2016;61:668-679.
11. Fuchs-Tarlovsky V, Rivera MA, Altamirano KA, Lopez-Al-
varenga JC, Ceballos-Reyes GM. Antioxidant supplementa-
tion has a positive effect on oxidative stress and hematological
toxicity during oncology treatment in cervical cancer patients.
Support Care Cancer. 2013;21:1359-1363.
12. Kang KM, Kang YN, Choi IB, et al. Effects of drinking hy-
drogen-rich water on the quality of life of patients treated with
radiotherapy for liver tumors. Med Gas Res. 2011;1:11.
13. Inal ME, Kanbak G, Sunal E. Antioxidant enzyme activities
and malondialdehyde levels related to aging. Clin Chim Acta.
2001;305:75-80.
14. Casado Á, Castellanos A, López-Fernández ME, et al. Deter-
mination of oxidative and occupational stress in palliative care
workers. Clin Chem Lab Med. 2011;49:471-477.
15.
Ishihara I, Nakano M, Ikushima M, et al. Effect of work conditions
and work environments on the formation of 8-OH-dG in nurses
and non-nurse female workers. J UOEH. 2008;30:293-308.
16. Fukuda S, Nojima J, Motoki Y, et al. A potential biomarker for
fatigue: Oxidative stress and anti-oxidative activity. Biol Psy-
chol. 2016;118:88-93.
17. Ostojic SM, Stojanovic MD. Hydrogen-rich water affected
blood alkalinity in physically active men. Res Sports Med.
2014;22:49-60.
18. Chalder T, Berelowitz G, Pawlikowska T, et al. Development of
a fatigue scale. J Psychosom Res. 1993;37:147-153.
19. Fukuda S, Takashima S, Iwase M, Yamaguchi K, Kuratsune
H, Watanabe Y. Development and validation of a new fatigue
scale for fatigued subjects with and without chronic fatigue
syndrome. In: Watanabe Y, Evengård B, Natelson BH, Jason
LA, Kuratsune H, eds. Fatigue Science for Human Health.
New York: Springer. 2008:89-102.
20. Kessler RC, Andrews G, Colpe LJ, et al. Short screening scales
to monitor population prevalences and trends in non-specic
psychological distress. Psychol Med. 2002;32:959-976.
21. Radloff LS. The CES-D scale: a self-report depression scale
for research in the general population. Appl Psychol Meas.
1977;1:385-401.
22.
Buysse DJ, Reynolds CF 3
rd
, Monk TH, Berman SR, Kupfer DJ.
The Pittsburgh Sleep Quality Index: a new instrument for psychi-
atric practice and research. Psychiatry Res. 1989;28:193-213.
23. Johns MW. A new method for measuring daytime sleepiness:
the Epworth sleepiness scale. Sleep. 1991;14:540-545.
24.
Doi Y, Minowa M, Uchiyama M, et al. Psychometric assess-
ment of subjective sleep quality using the Japanese version of
the Pittsburgh Sleep Quality Index (PSQI-J) in psychiatric dis-
ordered and control subjects. Psychiatry Res. 2000;97:165-172.
25. Furukawa TA, Kawakami N, Saitoh M, et al. The perfor-
mance of the Japanese version of the K6 and K10 in the World
Mental Health Survey Japan. Int J Methods Psychiatr Res.
2008;17:152-158.
26. Takegami M, Suzukamo Y, Wakita T, et al. Development of a
Japanese version of the Epworth Sleepiness Scale (JESS) based
on item response theory. Sleep Med. 2009;10:556-565.
27. Tanaka M, Fukuda S, Mizuno K, et al. Reliability and validity
of the Japanese version of the Chalder Fatigue Scale among
youth in Japan. Psychol Rep. 2008;103:682-690.
28. Shima S, Shikano T, Kitamura T. New self-rating scales for de-
pression. Clin Psychiatry. 1985;27:717-723.
29. Kanaya N, Hirata N, Kurosawa S, Nakayama M, Namiki A.
Differential effects of propofol and sevourane on heart rate
variability. Anesthesiology. 2003;98:34-40.
30. Takusagawa M, Komori S, Umetani K, et al. Alterations of
autonomic nervous activity in recurrence of variant angina.
Heart. 1999;82:75-81.
31. Akselrod S, Gordon D, Ubel FA, Shannon DC, Berger AC,
Cohen RJ. Power spectrum analysis of heart rate uctuation: a
quantitative probe of beat-to-beat cardiovascular control. Sci-
ence. 1981;213:220-222.
32. Pomeranz B, Macaulay RJ, Caudill MA, et al. Assessment of
autonomic function in humans by heart rate spectral analysis.
Am J Physiol. 1985;248:H151-153.
33. Malliani A, Pagani M, Lombardi F, Cerutti S. Cardiovascular
neural regulation explored in the frequency domain. Circula-
tion. 1991;84:482-492.
34. Appel ML, Berger RD, Saul JP, Smith JM, Cohen RJ. Beat to
beat variability in cardiovascular variables: noise or music? J
Am Coll Cardiol. 1989;14:1139-1148.
35. Tanaka M, Tajima S, Mizuno K, et al. Frontier studies on fa-
tigue, autonomic nerve dysfunction, and sleep-rhythm disorder.
J Physiol Sci. 2015;65:483-498.
36.
Montano N, Porta A, Cogliati C, et al. Heart rate variability ex-
plored in the frequency domain: a tool to investigate the link be-
tween heart and behavior. Neurosci Biobehav Rev. 2009;33:71-80.
37. Perini R, Veicsteinas A. Heart rate variability and autonomic
activity at rest and during exercise in various physiological
conditions. Eur J Appl Physiol. 2003;90:317-325.
38.
Tajima K, Tanaka M, Mizuno K, Okada N, Rokushima K, Wata-
nabe Y. Effects of bathing in micro-bubbles on recovery from
moderate mental fatigue. Ergonomia IJE&HF. 2008;30:134-145.
39. Mizuno K, Tanaka M, Tajima K, Okada N, Rokushima K,
Watanabe Y. Effects of mild-stream bathing on recovery from
mental fatigue. Med Sci Monit. 2010;16:Cr8-14.

Medical Gas Research ¦ December ¦ Volume 7 ¦ Issue 4 255
Mizuno et al. / Med Gas Res www.medgasres.com
40. Mizuno K, Tanaka M, Yamaguti K, Kajimoto O, Kuratsune
H, Watanabe Y. Mental fatigue caused by prolonged cognitive
load associated with sympathetic hyperactivity. Behav Brain
Funct. 2011;7:17.
41. Heart rate variability: standards of measurement, physiological
interpretation and clinical use. Task Force of the European So-
ciety of Cardiology and the North American Society of Pacing
and Electrophysiology. Circulation. 1996;93:1043-1065.
42. Kume S, Nishimura Y, Mizuno K, et al. Music improves sub-
jective feelings leading to cardiac autonomic nervous modula-
tion: a pilot study. Front Neurosci. 2017;11:108.
43. Kajimoto O. Development of a method of evaluation of fatigue
and its economic impacts. In: Watanabe Y, Evengård B, Na-
telson BH, Jason LA, Kuratsune H, eds. Fatigue Science for
Human Health. New York: Springer. 2008:33-46.
44. Mizuno K, Watanabe Y. Neurocognitive impairment in child-
hood chronic fatigue syndrome. Front Physiol. 2013;4:87.
45. Mizuno K, Tanaka M, Fukuda S, Imai-Matsumura K, Watanabe
Y. Relationship between cognitive functions and prevalence of
fatigue in elementary and junior high school students. Brain
Dev. 2011;33:470-479.
46. Kawatani J, Mizuno K, Shiraishi S, et al. Cognitive dysfunction
and mental fatigue in childhood chronic fatigue syndrome--a
6-month follow-up study. Brain Dev. 2011;33:832-841.
47. Mizuno K, Tanaka M, Fukuda S, Sasabe T, Imai-Matsumura
K, Watanabe Y. Changes in cognitive functions of students in
the transitional period from elementary school to junior high
school. Brain Dev. 2011;33:412-420.
48. Trotti R, Carratelli M, Barbieri M. Performance and clinical
application of a new, fast method for the detection of hydroper-
oxides in serum. Panminerva Med. 2002;44:37-40.
49. Nojima J, Motoki Y, Tsuneoka H, et al. 'Oxidation stress index'
as a possible clinical marker for the evaluation of non-Hodgkin
lymphoma. Br J Haematol. 2011;155:528-530.
50. Li J, Wang C, Zhang JH, Cai JM, Cao YP, Sun XJ. Hydro-
gen-rich saline improves memory function in a rat model of
amyloid-beta-induced Alzheimer's disease by reduction of oxi-
dative stress. Brain Res. 2010;1328:152-161.
51. Matsumoto A, Yamafuji M, Tachibana T, Nakabeppu Y, Noda
M, Nakaya H. Oral 'hydrogen water' induces neuroprotective
ghrelin secretion in mice. Sci Rep. 2013;3:3273.
52. Tomofuji T, Kawabata Y, Kasuyama K, et al. Effects of hydro-
gen-rich water on aging periodontal tissues in rats. Sci Rep.
2014;4:5534.
53. Watanabe Y, Kuratsune H, Kajimoto O. Desmond Biochemical
indices of fatigue for anti-fatigue strategies and products. In:
Matthews G, Desmond PA, Neubauer C, Hancoc PA, eds. The
Handbook of Operator Fatigue. CRC Press. 2012:209-224.
54.
Ataka S, Tanaka M, Nozaki S, et al. Effects of Applephenon and
ascorbic acid on physical fatigue. Nutrition. 2007;23:419-423.
55.
Mizuno K, Tanaka M, Nozaki S, et al. Antifatigue effects of coen-
zyme Q10 during physical fatigue. Nutrition. 2008;24:293-299.
56. Mizuma H, Tanaka M, Nozaki S, et al. Daily oral administra-
tion of crocetin attenuates physical fatigue in human subjects.
Nutr Res. 2009;29:145-150.
57. García-Niño WR, Zatarain-Barrón ZL, Hernández-Pando R,
Vega-Garcia CC, Tapia E, Pedraza-Chaverri J. Oxidative stress
markers and histological analysis in diverse organs from rats
treated with a hepatotoxic dose of Cr(VI): effect of curcumin.
Biol Trace Elem Res. 2015;167:130-145.
58. Atmaca M, Tezcan E, Kuloglu M, Ustundag B, Tunckol H. An-
tioxidant enzyme and malondialdehyde values in social phobia
before and after citalopram treatment. Eur Arch Psychiatry Clin
Neurosci. 2004;254:231-235.
59. Atmaca M, Kuloglu M, Tezcan E, Ustundag B. Antioxidant en-
zyme and malondialdehyde levels in patients with social pho-
bia. Psychiatry Res. 2008;159:95-100.
60. Maurya PK, Noto C, Rizzo LB, et al. The role of oxidative
and nitrosative stress in accelerated aging and major depres-
sive disorder. Prog Neuropsychopharmacol Biol Psychiatry.
2016;65:134-144.
61. Arranz L, Guayerbas N, De la Fuente M. Impairment of sev-
eral immune functions in anxious women. J Psychosom Res.
2007;62:1-8.
62.
Bouayed J, Rammal H, Younos C, Soulimani R. Positive corre-
lation between peripheral blood granulocyte oxidative status and
level of anxiety in mice. Eur J Pharmacol. 2007;564:146-149.
63. Pandya CD, Howell KR, Pillai A. Antioxidants as potential
therapeutics for neuropsychiatric disorders. Prog Neuropsycho-
pharmacol Biol Psychiatry. 2013;46:214-223.
64. Nakatomi Y, Mizuno K, Ishii A, et al. Neuroinammation in
patients with chronic fatigue syndrome/myalgic encephalo-
myelitis: an (1)(1)C-(R)-PK11195 PET study. J Nucl Med.
2014;55:945-950.
65. Castanon N, Luheshi G, Laye S. Role of neuroinammation
in the emotional and cognitive alterations displayed by animal
models of obesity. Front Neurosci. 2015;9:229.
66. Salim S, Chugh G, Asghar M. Inammation in anxiety. Adv
Protein Chem Struct Biol. 2012;88:1-25.
67. Liu L, Mills PJ, Rissling M, et al. Fatigue and sleep quality
are associated with changes in inammatory markers in breast
cancer patients undergoing chemotherapy. Brain Behav Immun.
2012;26:706-713.
68. Tian Y, Guo S, Zhang Y, Xu Y, Zhao P, Zhao X. Effects of hy-
drogen-rich saline on hepatectomy-induced postoperative cog-
nitive dysfunction in old mice. Mol Neurobiol. 2017;54:2579-
2584.
69. Johnson AW, Jaaro-Peled H, Shahani N, et al. Cognitive and
motivational decits together with prefrontal oxidative stress
in a mouse model for neuropsychiatric illness. Proc Natl Acad
Sci USA. 2013;110:12462-12467.
70. Bierhaus A, Wolf J, Andrassy M, et al. A mechanism converting
psychosocial stress into mononuclear cell activation. Proc Natl
Acad Sci USA. 2003;100:1920-1925.
71. Epel ES, Blackburn EH, Lin J, et al. Accelerated telomere
shortening in response to life stress. Proc Natl Acad Sci USA.
2004;101:17312-17315.
72.
Steptoe A, Hamer M, Chida Y. The effects of acute psychological
stress on circulating inammatory factors in humans: a review and
meta-analysis. Brain Behav Immun. 2007;21:901-912.
73.
Mizuno K, Tajima K, Watanabe Y, Kuratsune H. Fatigue correlates
with the decrease in parasympathetic sinus modulation induced by
a cognitive challenge. Behav Brain Funct.
2014;10:25.
74. Yamaguti K, Tajima S, Kuratsune H. Autonomic dysfunction in
chronic fatigue syndrome. Adv Neuroimmune Biol. 2013;4:281-
289.