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OPEN

Evaluation of the benefitsof neutral bicarbonate ionizedwater baths in an open‑label,randomized, crossover trial
Ryoko Ushikoshi‑Nakayama 1, Tomoe Yamazaki 1, Daisuke Omagari 1, Naoyuki Matsumoto 1,
Hiroko Inoue 2, Chiyoko Nukuzuma 3, Seiji Nishino 4 & Ichiro Saito 1,5*
We previously demonstrated that neutral bicarbonate ionized water (NBIW) bathing enhances bloodflow by bicarbonate ions and described the underlying mechanism. However, additional clinicalinvestigation was warranted to investigate the efficacy of NBIW bathing. Hence, we performed arandomized, open-label, crossover trial to examine the effects of NBIW bathing on mental stress,
sleep, and immune function. Participants who regularly felt stressed were randomly assigned to NBIW
or regular bathing for 4 weeks. Mental stress was assessed with the Brief Job Stress Questionnaire(BJSQ) and the Profile of Mood States Second Edition; sleep quality, with the Pittsburgh Sleep Quality
Index Japanese version (PSQI-J) and actigraphy; and immune function, with laboratory tests. PSQI-J
scores and actigraphy sleep latency and bed out latency improved significantly more with NBIW
bathing than with regular bathing (p < 0.05). Furthermore, NBIW bathing reduced both stress-induced
fluctuations in ­CD4+ and ­CD8+ T cell counts and fluctuations in the naive to memory T cell ratio andneutrophil phagocytosis, indicating improved immune function. These findings suggest that daily
NBIW bathing could improve mental stress, sleep quality, and immune function and bring about
positive health effects in those who experience stress in their daily lives.
Hot water balneotherapy in water rich in minerals and bicarbonate has traditionally been used around the­world1, and various studies have been performed to investigate the effectiveness of balneotherapy in t­ rauma2,
skin ­diseases3, and musculoskeletal d­ isorders4–7. In recent years, studies have reported positive effects of balneotherapy on sleep and metal ­stress4,8–10.
Mental stress was believed to affect biological homeostasis, such as emotional regulation and hormone secretion, and recently, it has become clear that it also causes sleep disorders, depression, and cardiovascular disease
and increases susceptibility to infectious diseases and c­ ancer11–13. In Japan, the morbidity rate due to mental
stress has been continuously increasing since the period of high economic growth in the late 1950s, with over
60% of employees reported to be experiencing mental anxiety and s­ tress14.
The suicide rate in Japan is by far the highest among the Group of Seven industrialized nations, and the leading
causes have been reported to include anxiety and depression related to mental stress in the ­workplace15. Despitea modest decrease in the number of suicides after the enactment of the Basic Act on Suicide Prevention by theJapanese Government in 2006, the annual number of suicides remains high at 20,00016. In 2015, the Japanese
Ministry of Health, Labour and Welfare mandated that workplaces conduct occupational stress checks on their­employees17,18. Nevertheless, the effectiveness of this measure is limited because of the small number of industrial
physicians affiliated with workplaces and the lack of methods for successfully coping with mental stress.
The COVID-19 pandemic deeply affected the lives of people globally, not only because of the physical healthrisks of infection, but also because of the considerable mental stress related to the significant pandemic-related
lifestyle changes, which in turn posed a threat to mental health and increased suicide r­ ates19. Against this1

Department of Pathology, Tsurumi University School of Dental Medicine, 2‑1‑3 Tsurumi, Tsurumi‑ku, Yokohama,
Kanagawa 230‑8501, Japan. 2Department of Pharmaceutical Sciences, Nihon Pharmaceutical University, 10281
Komuro, Ina‑machi, Kitaadachi‑gun, Saitama 362‑0806, Japan. 3Bicarbonate Thermotherapy Institute Co.,
Ltd., Park West Bldg. F7, 6‑12‑1 Nishishinjuku, Shinjuku‑ku, Tokyo 160‑0023, Japan. 4Psychiatry and Behavioral
Sciences, Sleep and Circadian Neurobiology Laboratory, Stanford University School of Medicine, 3155 Porter
Drive, Room 2016, Palo Alto, CA 94304, USA. 5Cranescience Co., Ltd., 3‑9‑8 Ginza, Chuo‑ku, Tokyo 104‑8139,
Japan. *email: saito@cranescience.co.jp
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background, the establishment of specific coping strategies to relieve stress is considered to be one of the mostcritical societal ­challenges20.
Previously, we performed a study on a neutral bicarbonate ionized water (NBIW) bath tablet. In contrast to
hot springs, in which the water composition varies depending on area and weather conditions, the quality ofour bathing tablet is stable, and people can enjoy balneotherapy at home without having to visit a hot spring.
Our previous study showed that bathing in NBIW tends to increase blood bicarbonate ion concentrations andincrease blood flow by increasing expression and phosphorylation levels of endothelial nitric oxide synthaseand levels of nitric oxide (NO) in femoral vascular tissue, effects that are associated with enhanced blood fl­ ow21.
Moreover, in a preliminary randomized controlled trial conducted on the basis of those experiments, we foundthat the effects of higher body temperature upon waking and one hour after bathing tended to occur earlier in
the intervention than in the control group. When a mood states profile was used to evaluate the effectiveness ofbathing in NBIW on stress improvement, vigor/activity scores were ­improved21. The earlier study did not evaluate
effects on immunity, but the positive effects of balneotherapy on immunity have received much attention in recent
years, and many studies have evaluated ­them6,7,22. Therefore, we conducted a randomized, open-label, crossoverstudy to investigate the effect of NBIW on sleep, mental stress, and immune function. In particular, to moreaccurately evaluate the efficacy of NBIW in improving mental stress, we assessed sleep quality, which correlateswith stress, not only subjectively with a questionnaire-based analysis, but also objectively with an activity meter.

Results

Study population

A flow diagram of the study population is shown in Fig. 1. Of 41 potential participants who understood thecontent of the study and provided written informed consent, 39 completed the screening test, which comprised a
simplified version of the Brief Job Stress Questionnaire (BJSQ) consisting of 23 ­questions23, the Pittsburgh Sleep
Quality Index Japanese version (PSQI-J)24, age, mean hours of sleep, height, weight, body mass index, bloodpressure, and pulse rate. Of those 39 individuals, 25 met the inclusion criteria, did not meet the exclusion criteria,
and exhibited no clinical abnormalities based on the results of the screening test, which included the results of
the BJSQ and the PSQI-J. This study used a crossover design comprising NBIW bath tablet bathing (NBIW) andstandard bathing (control). In one group, control bathing was conducted first (control-NBIW, n = 12), while in
the other group, NBIW bathing was conducted first (NBIW-control, n = 13). All 25 individuals participated inand completed the study and were eligible for inclusion in the efficacy analysis. Outcomes of each parameter were
measured before allocation and at completion of the intervention. After completion of the whole study period,a statistician independent from the study group performed the statistical analyses.

Background characteristics

Participant background characteristics are shown in Table 1. Analyses of background characteristics conductedwhen participants were allocated to groups after the screening test revealed no significant differences in age, mean
sleep duration, physical measurements, physiological tests, PSQI-J, or BJSQ between the 2 crossover groups, i.e.,
control-NBIW and NBIW-control. The percentage of NBIW tablets used (% ± SE) in relation to the number ofdays of use was not significantly different between the groups (control-NBIW, 99.70% ± 1.03%; NBIW-control,
100.00% ± 0.00%). Bathing time during the intervention was calculated from the diary data recorded by eachparticipant, and results confirmed that there was no significant difference in bathing time between NBIW bathing and control bathing.

Outcomes

Stress
The results of the BJSQ are shown in Fig. 2. Comparisons of percentage changes from baseline in 3 stressor categories (A, Job Stressors; B, Stress Reaction; and C, Social Support) revealed that the category C score decreased
significantly more with NBIW bathing than with control bathing (p = 0.0193, d = − 0.52 [medium]). There was asignificant difference in the BJSQ (category C score), one of the primary endpoints, between NBIW and control.
The sample size was confirmed post hoc: The sample size estimated by power analysis with the BJSQ category
C score was 25, and the power was 0.81. In addition, we confirmed that no significant carryover effect occurred(Fig. 3). Comparisons of the percentage change from baseline for each item in POMS2 are shown in Table 2.
The item Confusion-Bewilderment decreased significantly more with NBIW bathing. A decrease in values or“Improvement in stress” were observed in both interventions in many items of the primary and secondary
mental stress tests, but in both tests, the percentage changes were larger with the NBIW intervention than withthe control intervention.
Sleep
The baseline PSQI-J scores (mean ± SD) were 9.3 ± 1.8 in the control-NBIW group and 9.4 ± 1.4 in the NBIWcontrol group, i.e., in both groups, the scores indicated poor sleep quality (Table 1); however, with the NBIW
intervention, scores improved, i.e., decreased (to 5.8 ± 2.4; Supplementary Information 1), and the percentage change from baseline was significantly lower in the NBIW intervention than in the control intervention(p = 0.0238, d = − 0.42 [small]) (Fig. 4).
Sleep was also analyzed by actigraphy, which provided data on bedtime, total sleep time, sleep latency, wakeafter sleep onset, sleep efficiency, time out of bed, and bed out latency. We observed that over 4 weeks, NBIW
bathing generally improved sleep compared with control bathing. The results showed increased total sleep timeand sleep efficiency and decreased waking after sleep onset, sleep latency, and bed out latency, and paired t
tests showed that the decreases in sleep latency and bed out latency were significantly larger with the NBIW
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Enrollment
Assessed for eligibility (n = 41)
Healthy people aged 34–56 years
Excluded (n = 2)
Failure to complete screening test
Randomized(n = 39)
Met exclusion criteria (n = 14)

Assessment 1

Allocation

Group Control-NBIW (n = 12)Group NBIW-Control (n = 13)

Follow-Up
Pre-observation: 1 week(n = 12)Control bathing(n = 12)Pre-observation: 1 week(n = 13)

1st period
4 weeks

Assessment 2

NBIW bathing(n = 13)

Wash out: 1 week
NBIW bathing(n = 12)

2nd period
4 weeks

Assessment 3

Control bathing(n = 13)

AnalysisAnalyzed (n = 25)
Mean age, 49.4 ( 6.4) years; 10 men and 15
women
Figure 1.  Flow diagram of the randomized crossover-controlled trial. NBIW, neutral bicarbonate ionized water.
intervention than with the control intervention (Table 3). A power analysis of the items that showed significant
differences between the interventions determined that a sample size of 27 participants would be necessary,
assuming an effect size of 0.5, a probability of 0.05, and a power of 0.8. The post hoc calculations revealed larger
effect sizes for sleep latency (1.43) and bed out latency (1.59).

Immune functions

The results of the lymphocyte subset analyses are shown in Tables 4 and 5. The proportion of ­CD4+ cells, i.e.,
helper T cells, was significantly higher with the NBIW bathing intervention than with the control intervention(Table 4), whereas the concentration of ­CD8+ cells, i.e., cytotoxic T cells, was significantly lower with the NBIW
intervention than with the control intervention (Table 5). In comparison, in the control intervention, the proportion of ­CD4+ cells was significantly lower and the proportion of ­CD8+ and number of ­CD8+ ­CD28+ cells, which
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Mean (± SD)
Items

Group control-NBIW (n = 13)

Group NBIW-control (n = 12)

Between-group comparison, pvalue

Age (years)

49.3 ± 5.1

49.5 ± 7.6

0.6418Mean hours of sleep
Weekdays

5.67 ± 0.62

5.83 ± 1.11

0.2905

Weekends

6.38 ± 0.98

6.62 ± 1.02

0.5525Height (cm)

164.97 ± 10.28

162.65 ± 5.76

0.4888Weight (kg)57.78 ± 11.39

63.63 ± 13.51

0.2554

Body mass index (kg/m2)

21.02 ± 2.00

23.92 ± 4.12

0.0502

Systolic blood pressure (mm Hg)

112.6 ± 19.1

120.0 ± 8.7

0.2360

Diastolic blood pressure (mm Hg)

72.4 ± 14.3

74.8 ± 8.7

0.6106

Pulse rate (bpm)69.6 ± 9.9

72.0 ± 8.6

0.5214BJSQ score
A: Job stressors

18.3 ± 2.5

18.8 ± 2.9

0.6344

B: Stress reaction

25.0 ± 6.7

26.2 ± 5.2

0.6346

C: Social support

16.8 ± 2.6

16.5 ± 3.2

0.8025

PSQI-J total score

9.3 ± 1.8

9.4 ± 1.4

0.8332Bathing time during the intervention (min)NBIW (n = 25)

Control (n = 25)

22.8 ± 3.8

22.7 ± 3.5

0.8269

Table 1.  Background characteristics of the participants. Between-group comparisons of age, mean hoursof sleep, body mass index, and bathing time during the intervention were tested by Wilcoxon rank sum test;
height, weight, diastolic blood pressure, pulse rate, Brief Job Stress Questionnaire, and the Pittsburgh Sleep
Quality Index Japanese version, by Student’s t test; and systolic blood pressure, by Welch’s t test. BJSW, Brief
Job Stress Questionnaire; NBIW, neutral bicarbonate ionized water; PSQI-J, Japanese version of the Pittsburgh
Sleep Quality Index.

A: Job Stressors
Control

NBIW

#

#

B: Stress Reaction
Control

NBIW

C: Social Support
Control

NBIW

-5.00
-10.00-15.00-20.00-25.00-30.00#-35.00-40.00##*Percentage change from baseline

(%)
0.00

Figure 2.  Assessment of stress with the Brief Job Stress Questionnaire. The Brief Job Stress Questionnaire(BJSQ), simplified version for primary outcome of mental stress, consists of 23 questions and was scoredseparately for the 3 stress categories. The graph shows the rate of change in scores from baseline (minus SD)on the vertical axis. A: control, − 13.92 (− 14.13); NBIW, − 16.32 (− 11.92). B: control, − 18.96 (− 17.03);
NBIW, − 24.08 (− 10.53). C: control, − 4.14 (− 11.25); NBIW − 11.11 (− 15.08). N = 50 (control, n = 25; NBIW,
n = 25). *p < 0.05 tested by paired t test. #: Parameters with significant changes from baseline (p < 0.05; tested byWilcoxon rank sum test). NBIW, neutral bicarbonate ionized water.

represent the progenitor cells of C
­ D8+ cells, were significantly higher (Tables 4 and 5). Although there was nomarked change in ­CD4+ T cell counts in either intervention, C­ D8+ T cell counts showed a significant increase
with the control intervention (Table 5). These results indicate that the post-intervention C­ D4+ to C
­ D8+ T cellabundance ratio (­ CD4+:CD8+) was higher in the NBIW intervention (although the difference compared withthe control intervention was not significant) and showed a significant decrease with the control intervention(Table 5). ­CD4+CD45RA+, i.e., naive T cells, significantly decreased with the NBIW intervention (Table 4), resulting in a significant decrease in the ratio of the naive to memory T cells ratio (N:M ratio) by the NBIW intervention (Table 5). At Week 4, the N:M ratio was not significantly different between NBIW and control. However,when compared with the baseline, it was increased by the control intervention and significantly decreased by
the NBIW intervention (Table 5). The proportion of C
­ D16+CD56- cells, i.e., mature natural killer (NK) cells,
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p = 0.308
Percentage change from baseline
in C: Social Support

15.0

p = 0.335

10.0
5.0
0.0
-5.0
-10.0
-15.0
-20.0
-25.0
-30.0
-35.0

Control-NBIW NBIW-Control

Control-NBIW NBIW-Control

1st
period
1st period

2nd
period2nd period

Figure 3.  Test for carryover effect. For the percentage change from baseline in the Brief Job Stress
Questionnaire-Social Support (BJSQ-C), the intervention periods were grouped into NBIW and control andcompared with a t test. The first and second periods of the control intervention showed no significant difference(p = 0.308), and the same was found for the first and second periods of the NBIW intervention (p = 0.335). Thus,
no carryover effect was observed.

Percentage change from baseline

Within-group comparison
Between-group comparison

Items
AH (anger-hostility)%

p value

Control (n = 25), mean (± SD)

NBIW (n = 25), mean (± SD)

p valuer

Effect size

Control

NBIW− 8.0 ± 14.7

− 9.6 ± 12.8

0.3341− 0.14Small

0.0083**0.0014**CB (confusion-bewilderment)%− 3.2 ± 11.9

− 10.0 ± 11.6

0.0478*− 0.28Small

0.19670.0005**DD (depression-dejection)%− 6.1 ± 13.3

− 8.9 ± 12.1

0.2209− 0.17Small

0.0350*0.0024**

FI (fatigue-inertia)%− 7.5 ± 13.4

− 13.0 ± 11.5

0.1064− 0.23Small

0.0090**0.0001**TA (tension-anxiety)%− 11.6 ± 13.5

− 14.2 ± 9.8

0.3651− 0.13Small

0.0007**0.0000**VA (vigor-activity)%

15.1 ± 16.0

19.4 ± 21.4

0.3271− 0.14Small

0.0008**0.0009**F (friendliness)%

1.4 ± 18.0

9.2 ± 17.8

0.0674− 0.26Small

0.5988**0.0547

TMD (total mood disturbance)%− 10.4 ± 13.0

− 15.2 ± 9.9

0.0883− 0.24Small

0.0006**0.0000**

Table 2.  Assessment of sleep quality by the profile of mood states 2nd edition. Between-group and withingroup comparisons were tested by Wilcoxon signed-rank test. *p < 0.05, **p < 0.05. Effect size was calculated asr family: small (0.1 ≤|r|< 0.3), medium (0.3 ≤|r|< 0.5), large (0.5 ≤|r|). NBIW, neutral bicarbonate ionized water.

decreased significantly in the NBIW intervention. At Week 4, CD16+CD56− (%) was not significantly different
between NBIW and control. When compared with baseline, it decreased significantly in the NBIW intervention
but showed no significant decrease in the control intervention (Table 4). The proportion of ­CD20+ cells, i.e., B
cells, and the number of B cells increased significantly after both the NBIW and control interventions (Tables 4
and 5). The proportion of ­CD3+ cells, i.e., mature T cells, in total lymphocyte counts did not differ significantly
between the interventions. Neutrophil phagocytosis activity and NK cell activity increased significantly with bothinterventions. However, mean neutrophil phagocytosis activity at Week 4 was higher with the NBIW intervention
than with the control intervention. As a result, effects on neutrophil phagocytosis activity were greater with the
NBIW intervention than with the control intervention (Table 6).

Adverse events

A total of 10 adverse events were reported during the course of this study, 7 of which occurred during the control
intervention. No significant differences in the occurrence of adverse events were observed between the 2 interventions. In addition, reports of abdominal pain, diarrhea (loose stools), weight loss, and elevated creatinine
kinase during the use of NBIW were judged by the investigator to be not causally related to the study product.
Fisher’s exact test did not find a significant difference in the incidence of adverse events between NBIW andScientific Reports |(2024) 14:1261 |https://doi.org/10.1038/s41598-024-51851-9

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Percentage change from baseline
in PSQI-J(%)

Control

NBIW

Figure 4.  Sleep quality assessment with the Japanese version of the Pittsburgh Sleep Quality Index. The primary
outcome sleep quality was assessed by the Japanese version of the Pittsburgh Sleep Quality Index. The graphshows the percentage change in scores from baseline (minus SD) on the vertical axis. Control, − 25.67% (SD,− 27.67%); neutral bicarbonate ionized water, − 37.08% (SD, − 26.33%). *p < 0.05 tested by paired t test. NBIW,
neutral bicarbonate ionized water.

Items

Control (n = 25), mean ± SD

NBIW (n = 25), mean (± SD)

Total sleep time

318.44 ± 13.8

320.96 ± 13.47

Sleep latency

14.24 ± 1.68

12.06 ± 1.28

Wake time after sleep onset

72.24 ± 7.88

68.12 ± 5.80

Sleep efficiency

77.52 ± 1.84

78.85 ± 1.57

Sleep period time390.68 ± 16.57

389.08 ± 15.22

Time in bed

412.74 ± 16.89

407.57 ± 15.13

Bed out latency

7.82 ± 0.98

6.43 ± 0.71

Significant difference between groups**

Table 3.  Evaluation of sleep quality by actigraphy. Between-group comparisons were tested by a paired t test(one-tailed). NBIW, neutral bicarbonate ionized water. *p < 0.05.

control (P = 1.000, Effect size: φ = 0.0576). No severe or serious adverse events or adverse drug reactions wereobserved (Table 7).

Discussion

Balneotherapy, i.e., the treatment of diseases by bathing, has been practiced since ancient times for therapeuticand medical purposes, and its therapeutic effects on cardiovascular and dermatological diseases have been
­documented25–27. Our previous double-blind, placebo-controlled study of NBIW and our in vitro and in vivo
studies suggested that NBIW increases body temperature, promotes blood flow via nitric oxide production,and improves mental stress and sleep ­quality21. However, the placebo control contained magnesium sulfate andsodium sulfate, both of which are known to promote blood circulation; hence, warm bathing in the placebo
control also showed mild effects in some analysis items, making it difficult to analyze the effectiveness of NBIW.
Therefore, we conducted the present randomized, open-label, crossover trial to further investigate the effects ofbathing in NBIW. The study included assessments of immune function because of the close correlation between
stress, sleep, and immune function.
A number of previous studies reported on the efficacy of balneotherapy in improving mental stress and sleep
disorders, and a large-scale randomized controlled trial (n = 362) conducted in 2016 in Chongqing, China,
showed that balneotherapy is effective in reducing mental stress and sleep disorders and alleviating general health­concerns8. Moreover, given that balneotherapy reduces levels of cortisol, a stress biomarker, some authors havesuggested that it may be beneficial in controlling mental s­ tress28. In the stress assessment in the present study,
BJSQ categories A (Job Stressors) and B (Stress Reaction) were significantly improved after both the NBIW andcontrol interventions. On the other hand, category C (Social Support) was significantly improved by the NBIW
intervention but showed no change with the control intervention. For Category A and B, bathing itself is seen
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Between-group comparison (4 weeks)Control (n = 25), mean(± SD)
CD3+

%

CD4 +

%

CD4+CD45RA+

%

CD8+

%

CD8+CD28+%

CD16+CD56−%

CD16−CD56+%

CD16+CD56+

%

CD20+

%

Baseline

73.936 ± 6.476

Week 472.460 ± 6.052

Baseline

51.861 ± 7.358

Week 450.025 ± 6.382

Baseline

43.172 ± 11.520

Week 443.142 ± 12.323

Baseline

12.326 ± 5.408

Week 413.382 ± 5.743

Baseline

73.261 ± 12.081

Week 472.256 ± 13.544

Baseline

0.719 ± 0.431

Week 4

0.587 ± 0.405

Baseline

4.456 ± 3.128

Week 44.570 ± 3.418

Baseline

7.868 ± 3.198

Week 48.472 ± 2.885

Baseline

15.623 ± 4.726

Week 4

16.698 ± 5.421

NBIW (n = 25), mean(± SD)
72.998 ± 6.561
51.563 ± 7.453
41.448 ± 11.545
12.416 ± 5.927
72.314 ± 12.797
0.500 ± 0.355
4.477 ± 2.966
8.008 ± 2.993
16.630 ± 5.203

Within-groupcomparisonppd or rEffect size

Control

NBIW

0.36330.09–

0.0044**0.08340.0395*

0.22

Small

0.0076**

0.65140.1166− 0.14–

0.97460.0062**0.1815− 0.17–0.0065**0.8746

0.7982− 0.04–0.2641

0.49260.1424− 0.21Small

0.19350.0017**0.8823− 0.02–0.76720.38170.3958− 0.16–0.2406

0.79950.8625− 0.01–0.0366*0.0222*

Table 4.  Effect of the intervention on lymphocyte subset percentages. Distribution of CD3+, CD4+,
CD4+CD45RA+, CD8+, CD16+CD56+, and CD20+ populations was parametric, and distribution of otherpopulations was non-parametric. Between-group and within-group comparisons were tested by a pairedt test (for parametric data) and Wilcoxon signed-rank test (for non-parametric data). *p < 0.05, **p < 0.01.
For parametric data, effect size was calculated as Cohen’s d: small (0.2 ≤|d|< 0.5), medium (0.5 ≤|d|< 0.8),
large (0.8 ≤|d|); for non-parametric data, effect size was calculated as r family: small (0.1 ≤|r|< 0.3), medium(0.3 ≤|r|< 0.5), large (0.5 ≤|r|). NBIW, neutral bicarbonate ionized water.

to be effective in reducing stress. Because there was a significant improvement also in category C with NBIW
bathing compared with standard bathing without any additions, NBIW bathing is considered to be more effective in reducing stress. Moreover, the questions related to BJSQ category C evaluate whether a study participant
receives sufficient support from those around them. We suggest that NBIW bathing improved study participants’mental condition, which resulted in a change in their interpersonal cognition.
Balneotherapy over a period of 2 to 3 weeks may have beneficial effects on sleep ­quality29. It is thought toimprove sleep by lowering systemic blood pressure and core body temperature by dilating peripheral blood vessels throughout the body, reducing pain by inhibiting inflammation and pain-related substances, and relaxing­muscles30. The decrease in blood pressure occurs when the parasympathetic nervous system is dominant. Dominance of the parasympathetic nervous system is also considered to improve sleep quality. In fact, percutaneous
stimulation of the parasympathetic nerve was reported to improve sleep quality in retired veterans suffering frompost-traumatic stress d­ isorder31. The PSQI-J, one of the primary sleep endpoints in this study, assesses subjective sleep quality, including insomnia; the total score ranges from 0 to 21, and a score of 6 or more indicates apotential sleep p­ roblem24,32. In the present study, the PSQI-J score confirmed that the NBIW intervention led
to significantly greater improvements in subjective sleep quality than the control intervention. Furthermore, in
the actigraphy sleep assessment, which was used as an objective assessment of sleep, the mean values of each
item during the 3-week period showed a significant reduction in sleep latency and bed out latency and a trend
towards improved sleep in many items with the NBIW intervention compared with the control intervention.
Mental stress and sleep quality are known to interact closely, and studies have shown that sleep quality is
reduced in stressful ­environments33,34 and that stress can be reduced by improving sleep ­quality35. In this study,
NBIW bathing was considered to improve the quality of sleep not only subjectively but also objectively. In ourprevious study, we demonstrated that the bicarbonate ions in NBIW affect endothelial cells, and through phosphorylation of endothelial nitric oxide synthase, promote synthesis of nitric oxide, which dilates blood vessels,leading to improved blood flow and temperature elevation. Moreover, 4-week NBIW bathing improved sleep
quality according to PSQ-J score and reduced stress according to POMS2 s­ cores21. Together with the result of
this study, these findings indicate that NBIW may improve sleep quality and decrease stress by promoting blood
circulation via production of nitric oxide and increase of body temperature.
A large number of studies have shown that mental stress increases the risk of a wide range of diseases andmay be a risk factor for cancer and autoimmune ­diseases36,37. Mental stress is also particularly strongly associatedwith cardiovascular ­disease38. For example, several articles have reported that mental stress induces myocardial ischemia in patients with coronary artery ­disease39,40 and that depression is correlated with cardiovascular­disease41. In addition, the Japan Collaborative Cohort Study for Evaluation of Cancer Risk (JACC study), which

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Between-group comparison (week4)
Control (n = 25), mean(± SD)
Number of T cells/µL

Number of ­CD4+ T cells/µL

Number of ­CD8+ T cells/µL

CD4+:CD8+ ratio–

Number of naive T cells/µL

Number of memory T cells/µL

N:M ratio–

Number of ­CD8+CD28+ T cells/µL

Number of B cells/µL

Number of natural killer cells/µL

Baseline

1154.2 ± 281.7

Week 41203.0 ± 328.3

Baseline

805.4 ± 200.5

Week 4819.0 ± 179.4

Baseline

198.0 ± 111.6

Week 4230.2 ± 131.3

Baseline

5.209 ± 2.667

Week 44.800 ± 2.985

Baseline

349.4 ± 142.5

Week 4355.0 ± 139.2

Baseline

456.0 ± 131.6

Week 4464.0 ± 131.0

Baseline

0.826 ± 0.354

Week 40.833 ± 0.375

Baseline

145.8 ± 94.6

Week 4168.6 ± 114.2

Baseline

251.3 ± 114.8

Week 4283.6 ± 133.8

Baseline

125.5 ± 69.6

Week 4142.4 ± 69.2

NBIW (n = 25), mean(± SD)
1177.6 ± 352.9
830.6 ± 266.8
204.7 ± 123.3
5.935 ± 4.399
343.5 ± 166.7
487.2 ± 171.4
0.770 ± 0.334
147.2 ± 99.7
268.2 ± 107.8
130.4 ± 74.3

Within-groupcomparisonppd or rEffect size

Control

NBIW

0.3065− 0.14Small

0.4926

0.98930.8191− 0.03–0.74680.70640.0395*− 0.29Small

0.0011**0.76720.1829− 0.19Small

0.0110*0.92500.0614− 0.26Small

0.84010.19190.35290.15–

0.59350.10650.1253− 0.18–0.85780.0070**0.0561− 0.27Small

0.0071**

0.79820.1918− 0.18Small

0.0061**0.0333*0.2108− 0.18Small

0.09790.3531

Table 5.  Effect of the intervention on lymphocyte subset concentrations. The distribution of memory T cell
population and naive to memory T cells ratio (N:M ratio) were parametric; distribution of other populations
was non-parametric. Between-group and within-group comparisons were tested by paired t test (forparametric data) and Wilcoxon signed-rank test (for non-parametric data). *p < 0.05, **p < 0.01. Effect size
was calculated for parametric data as Cohen’s d: small (0.2 ≤|d|< 0.5), medium (0.5 ≤|d|< 0.8), large (0.8 ≤|d|),for non-parametric as r family: small (0.1 ≤|r|< 0.3), medium (0.3 ≤|r|< 0.5), large (0.5 ≤|r|). NBIW, neutral
bicarbonate ionized water; N:M ratio, ratio of naive to memory T cells.

Between-group comparison (week 4)
Control (n = 25), mean(± SD)
Neutrophil phagocytosis
activity%

NK cell activity%

Baseline

83.4 ± 2.4

Week 484.1 ± 2.0

Baseline

24.9 ± 13.8

Week 435.7 ± 11.8

NBIW (n = 25), mean(± SD)
84.6 ± 2.2
34.9 ± 11.4

Within-groupcomparisonpprEffect size

Control

NBIW

0.1542− 0.20Small

0.0350*0.0044**0.7378− 0.05–0.0000**0.0001**

Table 6.  Neutrophil phagocytosis activity and natural killer cell activity. Between-group comparisonswere tested by Wilcoxon signed-rank test. *p < 0.05, **p < 0.01. Effect size was calculated as r family: small(0.1 ≤|r|< 0.3), medium (0.3 ≤|r|< 0.5), large (0.5 ≤|r|). NBIW, neutral bicarbonate ionized water; NK, naturalkiller.

was sponsored by the Ministry of Education, Science, Sports and Culture of Japan, identified mental stress as
being associated with increased coronary artery disease and increased stroke mortality in w
­ omen42, and in thelarge-scale international case–control INTERHEART study, psychosocial factors were found to more than double the risk of myocardial i­ nfarction43. One of the primary mechanisms underlying the onset of cardiovasculardisease due to mental stress may be inflammation-mediated vascular endothelial dysfunction resulting from therelease of inflammatory cytokines as a result of suppression of the parasympathetic nervous ­system44. A previous
study showed that the bicarbonate ions in NBIW act directly on vascular endothelial cells to induce nitric oxide
production through phosphorylation of endothelial nitric oxide s­ ynthase21. As such, these findings suggest that
continued NBIW bathing with warm water may also reduce the risk of cardiovascular disease by improving bothmental stress and vascular endothelial function.
Immune function is also thought to be closely related to stress and s­ leep45,46. Several studies have shown thatstress is a risk factor for cancer and autoimmune diseases, suggesting that stress affects immune tolerance andScientific Reports |Vol:.(1234567890)(2024) 14:1261 |https://doi.org/10.1038/s41598-024-51851-98

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Item
NauseaSymptoms

Adverse events

Severity

Incidence

Adverse drug reactions

Control

NBIW

1

0

Malaise2

0

Headache10Skin reddening

10

Nasal discharge

10Abdominal pain

10

Abdominal pain, diarrhea (loose stool)

01

Weight loss

01

Increase in creatine kinase

0

1

Mild

4

3

Moderate00

Severe00Number of patients

4

3

Number of events

7

3

Symptoms

–

–

–

Mild

0

0

Severity

Moderate

00Incidence

Severe00Number of patients00

Number of events

0

0

Table 7.  Incidence of adverse events and adverse drug reactions. Significant values are in bold.

anticancer ­immunity37,47. Furthermore, sleep deprivation is known to alter the secretion of inflammatory markers such as interleukins, tumor necrosis factor-α, other cytokines, chemokines, and acute phase ­proteins48,49.
In this study, changes in several immune factors were observed with NBIW and control bathing. The­CD4+:CD8+ T cell ratio did not change significantly with the NBIW bathing intervention, but it decreased withthe control bathing intervention because of an increase in ­CD8+ T cells. ­CD8+ T cells are known to fluctuate
in number and function in response to s­ tress50–52. These suggest that the ­CD8+ T-cell count decreased because
NBIW bathing resulted in a decrease in stress.
The proportion of naive T cells, as represented by C
­ D4+CD45RA+, decreased slightly after NBIW bathing,while the number of memory T cells showed increase, resulting in a lower N:M ratio. Thus, given that tissueresident and circulating memory T cells play an essential role in anti-tumor immunity, NBIW bathing may
enhance anti-tumor immune f­ unction53.
The proportion of ­CD16+CD56- cells, a marker of mature NK cells, decreased significantly in the NBIW
bathing intervention. ­CD16+CD56- mature NK cells are increased by post-traumatic ­stress42,54, suggesting thatNBIW bathing may reduce C­ D16+CD56− mature NK cells by alleviating stress.
The proportion of C
­ D20+ B cell markers among total lymphocytes, the number of B cells, and the level ofneutrophil phagocytosis and NK cell activity increased significantly after both interventions, suggesting thatwarm bathing itself improves immune function. However, the mean value of neutrophil phagocytosis activity
after 4 weeks was higher with NBIW bathing than with control bathing, and the change was greater with NBIW
than with control.
This study has some limitations. First, most of the participants were middle aged. Second, people with selfperceived daily stress were recruited. Third, stress is subjective. These factors may limit the generalizability of
the results.
Taken together, the results of this study suggest that NBIW bathing has a positive influence on stress, sleep,and immune function. Stress, sleep, and immune function interact with each ­other45,46, so the effect of NBIW
on immune function may be mediated through the improvements in stress and sleep. In addition, given thatbicarbonate ions stimulate nitric oxide production in macrophage cell lines stimulated by lipopolysaccharide
and interferon-gamma, which in turn promotes inflammatory r­ esponses55,56, it is conceivable that increased
bicarbonate ions in the blood due to NBIW bathing may have a direct effect on immune system cells. In the
future, further studies of such mechanisms are warranted.

Materials and methods

Participant eligibility and recruitment

This randomized clinical trial was conducted and reported in compliance with the Declaration of Helsinki and
the CONSORT Statement, respectively. It was approved by the Chiyoda Paramedical Care Clinic Ethics Review
Board (IRB No.: 15000088; Approval No.: 22031805), and the protocol was registered with the UMIN-CTR(UMIN000047429) (07/04/2022). All participants provided written informed consent to participate in the study.
The study was conducted between April 13, 2022, and August 11, 2022, and no major changes were made to the

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protocol during the study. Data were collected at Chiyoda Paramedical Care Clinic and other institutions, andstatistical analyses were performed by the contract research organization, CPCC, Inc.
Participants were recruited by means of widespread dissemination of information at the study sites. Participation was voluntary. The inclusion criteria were as follows: (a) men and women aged 30 to 60 years at the time of
informed consent, (b) individuals who reported experiencing daily stress, (c) individuals who were dissatisfied
with their sleep, (d) women who were postmenopausal or had a regular menstrual cycle of 28 to 32 days, (e)individuals who were able to take a bath once a day during the study period, and (f) individuals who were able
to receive a full explanation of the study, understand its contents, and give their written consent. The exclusion
criteria included 14 items (see Supplementary Information 2) in consideration of their impact on the efficacy
evaluation and the safety of the participants. The major exclusion criteria included individuals taking medicinesor supplements that could potentially have an impact on the study; individuals with irregular lifestyle rhythms,
such as shift workers; individuals who may experience loss of consciousness due to seizures; individuals who
have experienced coarse oily skin due to bath salts; and individuals who had donated a certain amount of blood
before the start of the study. Study investigators and others enrolled study participants who gave written informed
consent, satisfied the inclusion criteria, did not meet the exclusion criteria, exhibited no clinical abnormalities
based on the results of the screening test, and were deemed eligible for participation in the study, taking into
account the results of the ­BJSQ23 and PSQI-J24. The allocation manager, who was independent of the study andanalysis sites, used stratified randomization to allocate eligible participants to 2 groups based on age, sex, and
BJSQ and PSQI-J scores at the time of the screening test. The parameters used for allocation were analyzed byan unpaired t test or a Wilcoxon rank sum test, and it was confirmed that there was no significant difference
between the two groups.
Subsequently, an allocation list was prepared with group names and participant identification numbers.

Study design and intervention

This study was designed as a randomized, open-label, crossover trial with 2 intervention periods, each lasting
4 weeks, with a 1-week washout period between. The duration of the study product use was set to 4 weeks because
another study showed an effect after 4 weeks of study product use, and the washout period was set to 1 week byreferring to that ­study21. The study findings at the start of the pre-observation period, 1 week before the start ofthe intervention, were used as the baseline data.
The study bath salts were NBIW tablets (HOTTAB Inc.) that were pH adjusted to release the maximum
amount of bicarbonate ions when dissolved in warm tap water. The composition of the tablets is shown in Table 8.
The tablets were white and weighed 15 g each; once participants had opened the package, they were asked tostore the tablets in a cool, dark place with low humidity.
Participants were divided into 2 groups: after the first 1-week pre-observation period, one group bathedwithout NBIW tablets (control-NBIW group) and the other bathed with NBIW tablets (NBIW-control group).
Then, after a 1-week washout period, each group participated in the other intervention. During the NBIW
bathing period, participants were instructed to add 4 NBIW tablets to their bath (amount of water: 180 L) oncea day and to enter the bath at least 15 min after adding the tablets. During the control period (including the preobservation and washout periods), no NBIW tablets or other bath products were to be added to the once-dailybaths. The bath water temperature was to be kept at 37 °C to 41 °C, and the bath was to be a full-body bath (i.e.,shoulder depth) lasting a minimum of 20 min. Participants were permitted to drink room temperature water
while bathing but were not permitted to drink cold water or read. The bath water was to be changed daily.

Outcome measures

Primary endpoints
Primary endpoints were mental stress assessed with the BJSQ (simplified version consisting of 23 ­questions23,
subjective sleep quality assessed with the PSQ-J24,32, and sleep measurements assessed with an activity meter(actigraphy)57,58. To obtain objective sleep variables, the small, lightweight waist-worn actigraphy device MTN221 (Acos, Co., Ltd.) was used. Participants wore this device on their waist all times except when bathing. Sleep
parameters were recorded for 3 weeks, from 5 days after the start of the intervention through to 2 days before its
completion. Sleep and wakefulness were analyzed by S­ leepSign®-Act Ver. 2.0 (Kissei Comtec Co., Ltd, Matsumoto,Japan), which relies on an algorithm that uses the activity and posture data recorded by the actigraphy device
in a series of linked c­ alculations59. This algorithm was used to evaluate bedtime, total sleep time, sleep latency,
wake after sleep onset, sleep efficiency, time out of bed, and bed out latency.

Ingredients%

Sodium bicarbonate

75

Sodium carbonate

5

Citric acid

15

Other

5

Table 8.  Ingredients of the neutral bicarbonate ionized water bath tablet.

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Secondary endpoints
POMS260,61 was evaluated as the secondary endpoint of mental stress. Among immunological tests, lymphocyte
subset, neutrophil phagocytosis, and NK cell activity were analyzed by flow cytometry (BD, FACS Caliber).
Interleukin (IL)-6, IL-8, and IL-12 in blood were measured by enzyme-linked immunosorbent assay (LBIS
Human IL-6 ELISA kit, LBIS Human IL-8 ELISA kit; FUJIFILM Wako Pure Chemical Corporation: Human
IL-12 ELISA kit; abcam).
The BJSQ, PSQ-J, and POMS2 were assessed at the time of screening and at the end of each intervention
period, and lymphocyte subset analyses were performed at the beginning of the pre-observation period and theend of each intervention period.
In addition, anthropometric measurements (height, weight, body mass index, abdominal circumference),
blood pressure and pulse rate, general hematologic tests, eosinophil count, and white blood cell counts were
performed. Furthermore, from the day after the start of the pre-observation period until the end of the second
intervention period, participants completed a daily log on a dedicated website that included information onwhether or not they had used the study product, whether or not they had taken baths, the duration of full-bodybathing, the time they went to bed and woke up, their diet, and their intake of medicines and supplements.

Statistical analysis

Microsoft Excel (Microsoft Corporation), IBM® SPSS26 (IBM Japan), BellCurve for Excel (Social Survey ResearchInformation Co. Ltd.), and G*Power 3.162 were used for tabulation, graphing, and analysis.
Data were analyzed with appropriate methods depending on normality, distribution, or correspondence. A
paired t test was used for parametric data, and Wilcoxon signed-rank test was used for non-parametric data.
In the case of parametric methods (paired t test), the effect size was calculated as Cohen’s d, and in the case of
non-parametric methods (Wilcoxon signed-rank test), it was calculated as r.

Data availability

The data used in this study are available from the corresponding author upon reasonable request.
Received: 4 July 2023; Accepted: 10 January 2024

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Acknowledgements

The study was funded by Cranescience Co., Ltd., Tokyo, Japan. Editing assistance was provided by Jacquie Klesing,Board-certified Editor in the Life Sciences (ELS), on behalf of Yamada Translation Bureau, Inc., Tokyo, Japan.

Author contributions

I.S. designed the study; T.Y., R.U.N., and S.N. contributed to the collection and analysis of study data; I.S., R.U.N.,
T.Y., D.O., N.M., H.I., C.N., and S.N. interpreted the data; and I.S., R.U.N., and S.N. wrote the article. All authors
approved the final manuscript after critical revision of the manuscript and agree to accept responsibility for its
scientific accuracy and consistency.
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Competing interests

IS a representative director of Cranescience Co., Ltd., Tokyo, Japan, and receives compensation and stock ownership. The other authors declare no competing interests.

Additional information

Supplementary Information The online version contains supplementary material available at https://​doi.​org/​
10.​1038/​s41598-​024-​51851-9.
Correspondence and requests for materials should be addressed to I.S.
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Scientific Reports |(2024) 14:1261 |https://doi.org/10.1038/s41598-024-51851-9

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