Previous studies investigating the influence of hot spring bathing on sleep quality have predominantly focused on the short-term effects basically depending on questionnaire without blood collection for biochemical tests. In this study, we undertook comprehensive investigation on the long-term health effects of hot spring bathing among the residents of Hot Spring Village and collected their blood samples for biochemical tests, including inflammatory cytokines (TNF-α) and neurotransmitters (5-HT and BDNF) analysis as well. We found that hot spring bathing (OR=0.18, 95%CI: 0.05-0.68), with the length of more than 30 minutes (OR=0.10, 95%CI: 0.02-0.53), and the frequency of more than 3 times per week (OR=0.07, 95%CI: 0.01-0.32) were protective factors for sleep quality (P<0.05). Interestingly, we observed at the first time that the blood TNF-α significantly decreased (P<0.05), with an increasing trend of 5-HT and BDNF in the bathing group. Besides, participants with good sleep quality exhibited significantly lower levels of TNF-α compared to those of poor ones, and among good sleepers aged 65 and older, higher levels of 5-HT were observed. Further logistic analysis revealed that a decrease of TNF-α (OR=1.03, 95% CI: 1.01-1.06) and an increase of 5-HT (OR=0.98, 95% CI: 0.97-0.99) were associated with good sleep quality. Additionally, the trends of decreasing TNF-α and increasing 5-HT were also
observed in the hot spring bathing group with good sleep quality for the first time. These findings suggested that hot spring bathing might improve sleep quality with the alteration of TNF-α and 5-HT, which could serve as potential indicators for future studies on health benefits of bathing.