OBJECTIVES: Healthy individuals have significant beat-to-beat variability in heart rate, and this variability decreases with mental stress. We aim to use heart rate variability (HRV) to objectively compare mental stress levels in otologic surgeons at rest and during key portions of procedures. DESIGN: Pilot study. SETTING: Operating room and laboratory. PARTICIPANTS: Two neurotology fellows performed six mastoidectomy and facial nerve (FN) dissections in the operating room and six in a cadaver lab while continuous electrocardiograms were measured wirelessly. Five-minute samples were recorded during resting, preoperative, mastoidectomy, and FN dissection. Beat-to-beat time intervals were analyzed in time and frequency domains. The standard deviation of normal beat-to-beat intervals (SDNN) and the ratio of low frequency to high frequency power (LF/HF, measure of sympathetic tone) were calculated. Decreases in SDNN and increases in LF/HF indicate elevated mental stress. RESULTS: Mean resting SDNN was 43.9 +/- 9.2 ms, not statistically different from preoperative SDNN (34.1 +/- 8.2 ms, p = 0.13). SDNN decreased during mastoidectomy (29.4 +/- 11.7 ms) and FN dissection (22.8 +/- 3.1 ms), which was significant compared to preoperative values (p = 0.03). Intraoperative LF/HF increased for FN dissection (6.8 +/- 2.6) compared to resting (2.2 +/- 0.7, p = 0.004), indicating increased sympathetic tone. Mastoid and FN cadaveric procedures resulted in SDNN of 33.6 +/- 3.8 and 32.9 +/- 4.7 ms, respectively, not statistically different from preoperative values (p = 0.82 and p = 0.94, respectively). Cadaveric FN dissection did not result in increased LF/HF (2.4 +/- 0.9) compared to resting (p = 0.94). CONCLUSIONS: Decreased HRV and increased sympathetic tone were observed intraoperatively, indicating high levels of mental stress, particularly with FN dissection. Similar changes were not found during cadaveric dissections.