Objective: To determine the influence of operating room familiarity on surgeon stress. Background: Regulating surgeon stress may improve patient safety. This study evaluated how assisting surgeon and operating room familiarity influence intraoperative heart rate variability among surgeons. Methods: Attending surgeons from seven specialties within four university hospitals in France were enrolled from 11/01/20-12/31/21. Vagal tone, an indicator of stress derived from heart rate variability, was assessed during the first five minutes after incision using the root mean square of successive differences (RMSSD). Higher RMSSD values indicate greater vagal tone. Team familiarity was quantified as the cumulative time the attending and assisting surgeons had operated together in the past, while operating rooms in which the surgeon conducted >10% of their operations were termed familiar. The effect of each on the RMSSD was assessed via a linear mixed-effect model adjusting for the random effect of the surgeon and possible confounders. Results: Overall, 643 surgeries performed by 37 surgeons were included. Median surgeon age was 49 years, 29(78.4%) were male, and 22(59.5%) were professors. Surgeons spent an average of 21.2 hours with the assisting surgeon prior to surgery and conducted 585(91.0%) of their operations in a familiar operating room. For every 10 additional hours spent operating together, ln(RMSSD) significantly increased by 0.018 (95%CI: 0.003 to 0.033, P =0.016). Familiar operating rooms also tended to increase surgeon ln(RMSSD) [0.098 (95%CI: -0.007 to 0.203, P =0.068)]. Conclusion: Familiar assisting surgeons, and potentially operating rooms, increased surgeon vagal tone. Maintaining a stable operating room environment may improve surgeon stress and patient care.