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Background: Physician burnout, characterized by chronic job-related stress leading to emotional exhaustion, depersonalization, and reduced personal accomplishment. This cross-sectional study investigates cortisol reactivity in male physicians with burnout compared to healthy controls during an acute psychosocial stress test. Methods: Sixty male physicians (30 burnout, 30 healthy controls) participated between September 2019 and December 2021 to investigate the impact of burnout on cardiovascular health. Salivary cortisol levels were measured before and after a Trier Social Stress Test (TSST). Burnout was assessed with the Maslach Burnout Inventory-Human Services Survey (MBI-HSS). Covariates included age, BMI, and physical activity. Data were analyzed using repeated measures analysis and area under the curve analysis. Results: Male physicians with burnout exhibited significantly greater cortisol reactivity during the TSST, notably post-stress to 15 min post-stress. Emotional exhaustion correlated with reduced cortisol increase from pre-stress and smaller post-stress to 15- and 45-min declines. Discussion: Findings suggest heightened cortisol reactivity in male physicians with burnout, possibly reflecting initial chronic stress stages. This study highlights the necessity for long-term research on cortisol’s influence on cardiovascular health and stress responses across diverse groups. Conclusions: The findings contribute to comprehending physiological responses in burnout-afflicted physicians, emphasizing cortisol reactivity’s pivotal role in stress-related research and its potential health implications, particularly within the burnout context.