Background Studies on a virtual reality simulator have demonstrated that sleep-deprived residents make more errors. Work-hour restrictions were implemented, among other reasons, to ensure enough sleep time for residents. The objective of this study was to assess the effects of sleep time, perceived fatigue, and experience on surgical performance. We hypothesized that performance would decrease with less sleep and fatigue, and that experienced surgeons would perform better than less experienced surgeons despite sleep deprivation and fatigue. Methods Twenty-two surgical residents and attendings performed a peg transfer task on two simulators: the Fundamentals of Laparoscopic Skills (FLS) trainer and the Virtual Basic Laparoscopic Surgical Trainer (VBLaST©), a virtual version of the FLS. Participants also completed questionnaires to assess their fatigue level and recent sleep hours. Each subject performed 10 trials on each simulator in a counterbalanced order. Performance was measured using the FLS normalized scores, and analyzed using a multiple regression model. Results The multiple regression analysis showed that sleep hours and perceived fatigue were not covariates. No correlation was found between experience level and sleep hours or fatigue. Sleep hours and fatigue did not appear to affect performance. Expertise level was the only significant determinant of performance in both FLS and VBLaST©. Conclusions Restricting resident work-hours was expected to result in less fatigue and better clinical performance. In our study, peg transfer task performance was not affected by sleep hours. Experience level was a significant indicator of performance. Further examination of the complex relationship between sleep hour, fatigue, and clinical performance is needed to support the practice of work-hour restriction for surgical residents.