BACKGROUND: Heart rate variability (HRV) has been used as a measure of stress and mental strain in surgeons. Low HRV has been associated with death and increased risk of cardiac events in the general population. The aim of this study was to clarify the effect of a 17-hour night shift on surgeons\' HRV. METHODS: Surgeons were monitored prospectively with an ambulatory electrocardiography device for 48 consecutive hours, beginning on a precall day and continuing through an on-call (17-h shift) day. We measured HRV by frequency domain parameters. RESULTS: We included 29 surgeons in our analysis. The median pulse rate was decreased precall (median 64, interquartile range [IQR] 56-70 beats per minute [bpm]) compared with on call (median 81, IQR 70-91 bpm, p < 0.001). Increased high-frequency (HF) activity was found precall (median 199, IQR 75-365 ms2) compared with on call (median 99, IQR 48-177 ms2, p < 0.001). The low-frequency:high-frequency (LF:HF) ratio was lower precall (median 2.7, IQR 1.9-3.9) than on call (median 4.9, IQR 3.7-6.5, p < 0.001). We found no correlation between the LF:HF ratio and performance in laparoscopic simulation. CONCLUSION: Surgeons working night shifts had a significant decrease in HRV and a significant increase in pulse rate, representing sympathetic dominance in the autonomic nervous system. TRIAL REGISTRATION: NCT01623674 (www.clinicaltrials.gov).