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Pilot study: Effects of drinking hydrogen-rich water on muscle fatigue caused by
acute exercise in elite athletes
Medical Gas Research 2012, 2:12 doi:10.1186/2045-9912-2-12
Kosuke Aoki (email@example.com)
Atsunori Nakao (firstname.lastname@example.org)
Takako Adachi (email@example.com)
Yasushi Matsui (firstname.lastname@example.org)
Shumpei Miyakawa (email@example.com)
Article type Research
Submission date 21 March 2012
Acceptance date 20 April 2012
Publication date 20 April 2012
Article URL http://www.medicalgasresearch.com/content/2/1/12
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Pilot study: Effects of drinking hydrogen-rich water
on muscle fatigue caused by acute exercise in elite
* Corresponding author
1 Doctoral Program in Sports Medicine, Graduate School of Comprehensive
Human Sciences, University of Tsukuba, Ibaraki, Japan
2 Department of Emergency and Critical Care Medicine, Hyogo College of
Medicine, 1-1, Mukogawa-cho, Nishinomiya, Hyogo 663-8501, Japan
Muscle contraction during short intervals of intense exercise causes oxidative stress, which
can play a role in the development of overtraining symptoms, including increased fatigue,
resulting in muscle microinjury or inflammation. Recently it has been said that hydrogen can
function as antioxidant, so we investigated the effect of hydrogen-rich water (HW) on
oxidative stress and muscle fatigue in response to acute exercise.
Ten male soccer players aged 20.9 ± 1.3 years old were subjected to exercise tests and blood
sampling. Each subject was examined twice in a crossover double-blind manner; they were
given either HW or placebo water (PW) for one week intervals. Subjects were requested to
use a cycle ergometer at a 75 % maximal oxygen uptake (VO2) for 30 min, followed by
measurement of peak torque and muscle activity throughout 100 repetitions of maximal
isokinetic knee extension. Oxidative stress markers and creatine kinase in the peripheral
blood were sequentially measured.
Although acute exercise resulted in an increase in blood lactate levels in the subjects given
PW, oral intake of HW prevented an elevation of blood lactate during heavy exercise. Peak
torque of PW significantly decreased during maximal isokinetic knee extension, suggesting
muscle fatigue, but peak torque of HW didn’t decrease at early phase. There was no
significant change in blood oxidative injury markers (d-ROMs and BAP) or creatine kinease
Adequate hydration with hydrogen-rich water pre-exercise reduced blood lactate levels and
improved exercise-induced decline of muscle function. Although further studies to elucidate
the exact mechanisms and the benefits are needed to be confirmed in larger series of studies,
these preliminary results may suggest that HW may be suitable hydration for athletes.
Since energy demands and oxygen consumption increase during supermaximal exercise, such
as intermittent running, sprints, and jumps, production of reactive oxygen species (ROS) and
reactive nitrogen species (RNS) also increase, threatening to disturb redox balance and cause
oxidative stress. During normal conditions, ROS and RNS are generated at a low rate and
subsequently eliminated by the antioxidant systems. However, a greatly increased rate of
ROS production may exceed the capacity of the cellular defense system. Consequently,
substantial free radicals’ attack on cell membranes may lead to a loss of cell viability and to
cell necrosis and could initiate the skeletal muscle damage and inflammation caused by
exhaustive exercise [1-3]. Although well-trained athletes suffer from less oxidative stress
reduction because their antioxidant systems adapt, accumulation of intense exercise can
provoke an increase in oxidative stress . To mitigate oxidative stress-induced adverse
events during sports, antioxidant supplementation among athletes has been well documented.
Although results of these studies are often contradictory depending on the antioxidant
compounds and quantity, some studies demonstrate the beneficial effects of antioxidants on
muscle fatigue or performance [5,6].
Recently, the beneficial effects of hydrogen-rich water (HW) have been described in
experimental and clinical disease conditions [7,8]. Although research on the health benefits of
HW is limited and there is scant data on long-term effects, pilot studies on humans suggest
that consuming HW may help prevent metabolic syndrome , diabetes mellitus , and
cancer patients’ side effects with radiotherapy . Since hydrogen is known to scavenge
toxic ROS  and induce a number of antioxidant proteins [13,14], we hypothesized that
drinking HW may be beneficial for athletes in reducing oxidative stress-induced muscle
fatigue following acute exercise. In this study, we evaluated the efficacy of hydrogen-rich
water on healthy subjects by measuring muscle fatigue and blood lactate levels after exercise.
Although further studies are needed to elucidate the exact mechanisms and benefits, this
report suggests that hydrogen-rich water might be an appropriate hydration fluid for athletes.
Ten male soccer players aged 20.9 ± 1.3 years old were subjected to exercise tests and blood
sampling. None of the subjects were smokers or were taking any supplements/medicines.
Each subject provided written informed consent before participation in accordance with the
University of Tsukuba’s Human Research Ethics Committee. Physical characteristics of the
subjects are shown in Table 1. All the players were involved in daily training sessions except
the day of experiment.
Table 1 Subjects’ Physical Characteristics (n
Age (year) 20.9
Height (cm) 172.0
Body weight (kg) 67.1
max (ml/kg/min) 53.2
BMI: body mass index, VO2max: maximal oxygen uptake
Generation of hydrogen-rich water
A plastic shelled product consisting of metallic magnesium (99.9 % pure) and natural stones
in polypropylene containers combined with ceramics (Doctor SUISOSUI®, Friendear,
Tokyo, Japan) was used to produce hydrogen. The product was capable of generating
hydrogen when placed in drinking water via the following chemical reaction:
Mg + 2H2O → Mg (OH)2 + H2. The magnesium stick or a placebo (a casting-only stick
without magnesium) was immersed in mineral water (Volvic®, Kirin Inc., Tokyo) for 24
hours prior to drinking. The final hydrogen concentrations of the placebo water (PW) and
hydrogen-rich water (HW) were 0 and 0.92 ~ 1.02 mM, respectively [9,11]. Each subject was
examined twice in a crossover double-blind manner, given either HW or PW for one week
Dose and mode of administration of hydrogen-rich water
Subjects were provided with three 500 ml bottles of drinking water and instructed to place
two magnesium sticks in each bottle 24 hours prior to drinking. Participants were asked to
drink one bottle at 10:00 PM of the day before the test, one at 5:00 AM, and one at 6:20 AM
on the day of examination. In summary, subjects consumed 1,500 ml of HW or PW.
The research protocol started at 6:00 AM. Subjects were given meals between 9:00 PM and
10:00 PM the day before experiments, and fasted overnight. No breakfast was given on the
day of the experiments. The subjects were first required to rest in a sitting position for 30
minutes. The exercise test consisted of the following: 1) Maximal progressive exercise test to
define maximal oxygen uptake (VO2max); 2) cycling an ergometer for 30 minutes at
approximately 75 % VO2max (Exercise-1); and 3) Running 100 maximal isokinetic knee
extensions at 90 ° sec-1 (Exercise-2). Blood samples were collected from an antecubital vein
just before Exercise-1 (6:30 AM), immediately after Exercise-1 (7:15 AM), immediately after
Exercise-2 (7:30 AM), 30 minutes after Exercise-2 (8:00 AM) and 60 minutes after Exercise-
2 (8:30 AM).
Maximal progressive exercise test
First, to define maximal oxygen uptake (VO2max), the subjects were subjected to a maximal
progressive exercise test on a bicycle ergometer (232CL, Conbiwellness, Tokyo). The test
consisted of a continuous step test beginning at a 30 W load, and increasing by 20 W every
minute until exhaustion. The subjects were instructed to ride at 50 rpm/min. Pulmonary gas
exchange values were measured using an exhaled gas sensor (AE280S, Minato Medical®,
Osaka, Japan) via a breath-by-breath system, and the mean values were calculated every 30
seconds for analysis. We determined that VO2max was reached when the oxygen
consumption reached its plateau .
Fixed-load cycling at 75 % (high intensity) of VO2 max
Before the test started, the subjects rested for two minutes. After warming up at a load of
50 W for one minute, the subjects were instructed to ride at submaximal levels for 30
minutes. Pulmonary gas exchange values were monitored to maintain VO2max at
approximately 75 %. During the experiments, the subjects were frequently verbally instructed
to control the range of motion to maintain VO2max at approximately 75 %.
Maximal isokinetic knee extensions
A calibrated Biodex System 3 isokinetic device (Biodex Medical Systems, New York, USA)
was used to measure peak torque (PT) and knee-joint position throughout 100 repetitions of
maximal isokinetic knee extension. During testing, each subject was seated on the Biodex
system 3 with 90 ° hip flexion, and restraining straps were placed across the waist and chest
in addition to a rigid sternal stabilizer. The dynamometer was motor driven at a constant
velocity of 90 °/sec. Each subject performed a series of 100 isokinetic contractions using the
knee extensors of the right leg from 90 ° of flexion to 0 ° (full extension). As the arm of the
dynamometer moved up from 90 ° to 0 °, subjects were encouraged to perform maximally for
each contraction throughout the full range of motion. Subjects relaxed as the dynamometer
arm moved back to 90 °. Each contraction and relaxation period lasted one second and the
total length of the contraction cycle was thus two seconds. All subjects were able to complete
the full 100 contractions.
Measurement of muscle fatigue
To measure muscle fatigue, the widely used First Fourier transform technique (FFT) is
utilized to analyze mean frequency of surface electromyogram (EMG) . EMG signals
were obtained from the rectus femoris muscle via electrodes connected to a 4-channel
frequency-modulation transmitter (Nihon Kohden, Tokyo, Japan). All data were stored and
analyzed using the FFT functions in Acknowledge 3.7.5 software (BIOPAC SYSTEM, Santa
Barbara, USA). Mean power frequency (MPF) and median power frequency (MDF) were
calculated as previously described . MPF shift of the EMG signal toward lower
frequencies has been extensively used in static contractions to indicate the development of
Blood lactate levels were determined using a commercially available Lactate Pro LT17170
kit (Arkray, Inc., Kyoto, Japan). The concentrations of derivatives of reactive oxidative
metabolites (dROMs) and biological antioxidant power (BAP) in the peripheral blood were
assessed using a Free Radical Analytical System (FRAS4; Wismerll, Tokyo, Japan).
Laboratory tests for creatine kinase (CK) were conducted using standardized procedures at
Kotobiken Medical Laboratory Services (Tokyo, Japan).
Repeated analysis of variance (ANOVA) tests were used to compare pre- and post-exercise
measurements. The F-test with Bonferroni post hoc group comparisons was performed where
appropriate. Probability values less than 0.05 were considered to be statistically significant.
SPSS 18.0 was used to perform the statistical analysis. Since the experiment was planned to
have a 90 % power of achieving significance at the 5 % level, the sample size in this model is
calculated to be between 8.91 and 9.25 (90 % power and 5 % significance level) in blood
lactate levels based on our previous experiences. Therefore, we assumed the sample size
would be appropriate for accumulation of preliminary data.
Blood analysis for lactic acid, d-ROMs, BAP and CK
As shown in Table 2, blood d-ROMs levels increased after exercise in subjects in both groups
treated with PW and HW. However, there was no statistical difference between the groups.
Eventhough the blood lactate level were significantly increased in both HW and PW at 45
and 60 min after exercise, these levels were comparably and significantly lower in the HW
than in the PW group (Figure 1).
Table 2 Changes in Blood Levels
0 min 45 min 60 min 90 min 120 min
(U.CARR) PW 269.0
(µmol/L) PW 2347.3
CK (IU/L) PW 247.0
Data were shown as mean ± standard deviation (SD). *p < 0.05 vs 0 min
Figure 1 Sequential changes of blood lactate levels during exercise. Blood lactate levels in
the athletes given PW significantly increased immediately after exercise compared to the
levels at pre-exercise. HW significantly reduced blood lactate levels post exercise using
bicycle ergometer. (*p < 0.05 vs. time 0. #p < 0.05 vs PW, N = 10)
Maximal knee extension exercise
At analysis for maximal knee extension exercise, we divided into five frames of 100-
repetition knee extension at the peak torque of isokinetic knee extension exercise . Each
frame was corresponded to 20 repetitions; Frame 1 for the first 20 repetitions, Frame 2 for the
following 21-40 repetitions, Frame 3 for 41-60 repetitions, Frame 4 for 61-80 repetitions and
Frame 5 for the last 81-100 repetitions. Although the peak torque of subjects treated with PW
significantly decreased during the first 40 repetitions (Frame 1-2), the reduction of peak
torque in the subjects given HW did not reach statistical difference, suggesting that HW
inhibited the early decrease of peak torque of the subjects.
MDF and MPF from EMG analysis
MDF and MPF in the subjects treated with PW or HW significantly decreased with time
during exercise. While these values significantly decreased at Frame 1-2, there was no
statistical difference between the subjects receiving PW and those receiving HW (Figure 2 B,
Figure 2 (A) Changes in Peak torque (PT) every 20 repetitions (rep
1 frame) during
100 maximum isokinetic knee extensions. PT of the subjects treated with PW significantly
decreased during the initial 40-60 contractions by approximately 20-25 % of the initial
values, followed by a phase with little change. On the other hand, there was no statistical
difference between Frame 1 and Frame 2 in HW, indicating that HW prevented the
decreasing the peak torque during the first 2 Frames. HW, Hydrogen rich water; PW, Placebo
water. (*p < 0.05 vs Frame 1, N = 10). (B) Changes in median frequency (MDF) every 20
1 Frame) during 100 maximum isokinetic knee extensions. Although
exercise significantly reduced MDF values during the first 2 Frames, there was no statistical
difference between HW and PW in all Frames. HW, Hydrogen rich water; PW, Placebo
water. (*p < 0.05 vs Frame 1, N = 10). (C) Changes in mean power frequency (MPF) every
20 repetitions (rep
1 Frame) during 100 maximum isokinetic knee extensions. There
was no statistical difference between HW and PW in all Frames. HW, Hydrogen rich water;
PW, Placebo water. (*p < 0.05 vs Frame 1, N = 10)
In this preliminary study, we showed that hydration with HW attenuated increase of blood
lactate levels and prevented post-exercise decrease of peak torque, an indicator of muscle
fatigue. Muscle fatigue is caused by many different mechanisms, including the accumulation
of metabolites within muscle fibers and the generation of an inadequate motor command in
the motor cortex. The accumulations of potassium, lactate, and H+ have often been suggested
as being responsible for the decrease in muscle contractility . In addition, aerobic,
anaerobic, or mixed exercise causes enhanced ROS production, resulting in inflammation and
cellular damage . Short bursts of heavy exercise may induce oxidative stress through
various pathways such as electron leakage within mitochondria, auto-oxidation of the
catecholamine, NADPH activity, or ischemia/reperfusion . Although the mechanism
involved in the efficacies of HW remains unclear, our results show that hydration with HW
could be feasible for acute exercise. Proper and adequate hydration is helpful for elite athletes
to achieve the best performance. HW can easily replace regular drinking water on a routine
basis and would potentially prevent adverse effects associated with heavy exercise.
Factors such as age, nutritional status, training level, and physical activity category can
influence the results [22,23]. Although we had anticipated that hydrogen, a known
antioxidant, would reduce oxidative stress following acute exercise, the effects of oral intake
of hydrogen rich water were marginal and did not affect the level of oxidative markers after
exercise. This can be explained by the facts that the athletes in our study have routinely
trained and their antioxidant defense systems may be more active. Previous studies reported
that repeated aerobic training increases antioxidant enzyme activity and subsequently
decreases oxidative stress [2,24-26]. Also, considering the short life-span of hydrogen in
circulation , more frequent drinking of HW during exercise might have additional effects.
In a future study, the efficacy of HW on untrained subjects or recreational exercisers, who
may have poorly established antioxidant systems to combat exercise-induced oxidative stress,
should be tested. Furthermore, different drinking protocols should be investigated.
We quantified muscle fatigue as a decline in the maximal force or power capacity of muscle,
which means that submaximal contractions can be sustained after the onset of muscle fatigue.
Similarly, blood lactate concentration is one of the most often measured parameters during
clinical exercise testing, as well as during performance testing of athletes. Lactate has often
been considered one of the major causes of both fatigue during exercise and post-exercise
muscle soreness. Lactate generated from the anaerobic breakdown of glycogen in the muscle
occurs only during short bouts of relatively high intensity exercise and it is usually related to
fatigue and muscle soreness. Previous evidence has shown that inorganic phosphate from
creatine phosphate was the main cause of muscle fatigue .
Dehydration in athletes may also lead to fatigue, poor performance, decreased coordination,
and muscle cramping. Although further investigations will be warranted, drinking HW may
be an appropriate hydration strategy . In this study, we administered HW or PW to
subjects prior to exercise. Further investigation is required to determine the best timing, dose,
and hydrogen concentration of drinking water to optimize the effects of HW.
In conclusion, our preliminary data demonstrated that consumption of HW reduced blood
lactate levels and improved muscle fatigue after acute exercise. Although further studies are
absolutely warranted, drinking HW would be a novel and effective fluid hydration strategy
The authors declare that they have no competing interests.
KA, TA and YM participated in the protocol design and the data accumulation. AN
conceived the study and drafted the manuscript. SM participated in the study design and
coordination. All authors read and approved the final manuscript.
This research was supported by a Daimaru Research Foundation grant awarded to SM.
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40 60 100 120
MPF (Hz) MDF (Hz) PT (N-M)
Frame 1 Frame 2 Frame 3 Frame 4 Frame 5