OBJECTIVE: This study evaluates the utility of serum s-αKlotho levels as a quantifiable biomarker for overwork. METHODS: Frontline medical workers aged 20-55 years from Yiling People's Hospital of Yichang were recruited. Criteria included nonsmokers, non-heavy drinkers, no chronic medication use, and no acute illnesses recently. Participants worked over 10 hours per day, 60 hours weekly, and had at least 3 years of experience. A control group was matched except for work conditions. Data were collected through surveys, and serum levels were measured. RESULTS: Significant differences in serum Klotho were found between overwork and control groups. The overwork group had higher median s-αKlotho levels (49.99 pg/mL) compared to controls (27.88 pg/mL). CONCLUSION: Overworked medical workers exhibited elevated serum s-αKlotho, suggesting s-αKlotho as a potential biomarker for overwork. Future research should use multicenter designs with larger samples to validate findings.