OBJECTIVE: To determine the specific effects of working long hours in surgery and potential cardiac stress in the individual surgeon by measuring heart rate variability (HRV). DESIGN, SETTING, AND PARTICIPANTS: This prospective study measured HRV before, during, and after a 24-hour shift in a standardized resting period of 10 minutes. Measurements were repeated over 10 shifts for each participant. Eight surgeons from a high-volume inner-city surgery department took part in the study. MAIN OUTCOME MEASURES: Time and frequency domain parameters of HRV as parameters of cardiac stress and correlations with perceived stress and fatigue on a visual analog scale. RESULTS: Perceived fatigue increased over 24 hours (P < .001), whereas stress levels decreased slightly (P = .06). Time domain parameters of HRV increased from before the shift to after the shift (standard deviation of normal to normal intervals, square root of the mean normal to normal interval, and percentage of adjacent pairs of normal to normal intervals differing by more than 50 milliseconds: all P < .01), denoting more cardiac relaxation. Both the low- and high-frequency components increased (P = .04 and P < .001, respectively), showing a heightened activity of the autonomic nervous system. CONCLUSIONS: Measurements of HRV during a 24-hour surgical shift did not show an increase in cardiac stress concerning time domain parameters despite intense workloads for a median of 20 hours. Frequency components increased in parallel, though, suggesting alterations in sympathovagal balance. Perceived stress levels correlated with HRV, whereas fatigue did not. Further studies on occupational stress and its cardiac effects in surgeons are needed.