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Objective: This study evaluates the utility of serum s-αKlotho levels as a quantifiable biomarker for overwork., Methods: Frontline medical workers aged 20-55 from Yiling People's Hospital of Yichang were recruited. Criteria included non-smokers, 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., Competing Interests: Conflict of Interest: NONE DECLARED, (Copyright © 2024 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American College of Occupational and Environmental Medicine.)