To assess the impact of a 3-hour polysomnography (PSG)-recorded night of sleep deprivation on next-morning simulated microsurgical skills among vitreoretinal (VR) surgeons with different levels of surgical experience and associate the sleep parameters obtained by PSG with Eyesi-generated performance. Self-controlled cohort study. Eleven junior VR surgery fellows with < 2 years of surgical experience and 11 senior surgeons with > 10 years of surgical practice. Surgical performance was assessed at 7am after a 3-hour sleep-deprived night using the Eyesi simulator and compared with each subject's baseline performance. Changes in Eyesi-generated score (0-700, worst to best), time for task completion (minutes), tremor-specific score (0-100, worst to best), and out-of-tolerance tremor percentage. Polysomnography was recorded during sleep deprivation. Novice surgeons had worse simulated surgical performance after sleep deprivation compared with self-controlled baseline dexterity in the total score (559.1 ± 39.3 vs. 593.8 ± 31.7; P= 0.041), time for task completion (13.59 ± 3.87 minutes vs. 10.96 ± 1.95 minutes; P= 0.027), tremor-specific score (53.8 ± 19.7 vs. 70.0 ± 15.3; P= 0.031), and out-of-tolerance tremor (37.7% ± 11.9% vs. 28.0% ± 9.2%; P= 0.031), whereas no performance differences were detected in those parameters among the senior surgeons before and after sleep deprivation (P ≥ 0.05). Time for task completion increased by 26% (P= 0.048) in the post-sleep deprivation simulation sessions for all participants with a high apnea-hypopnea index (AHI) and by 37% (P= 0.008) among surgeons with fragmented sleep compared with those with normal AHI and < 10 arousals per hour, respectively. Fragmented sleep was the only polysomnographic parameter associated with a worse Eyesi-generated score, with a 10% (P= 0.005) decrease the following morning. This study detected impaired simulated surgical dexterity among novice surgeons after acute sleep deprivation, whereas senior surgeons maintained their surgical performance, suggesting that the impact of poor sleep quality on surgical skills is offset by increased experience. When considering the 2 study groups together, sleep fragmentation and AHI were associated with jeopardized surgical performance after sleep deprivation. The authors have no proprietary or commercial interest in any materials discussed in this article.