Purpose: We aimed to investigate the effect of four distracting factors on surgical performance in virtual vitreoretinal surgery. Methods: Nineteen novice surgeons completed a validated training programme on the Eyesi surgical simulator (VRmagic GmbH, Manheim, Germany; software version 2.9.2) until a calculated target score was reached. The training programme consisted of four modules: navigation level 2 (Nav2), bimanual training level 3 (BimT3), posterior hyaloid level 3 (PostH3) and internal limiting membrane peeling level 3 (ILMP3). When properly trained, the participants completed the training programme once without distraction to generate reference scores and times. Next, they conducted the training programme under the influence of each of the following: auditory distraction, 12 hr of fasting, interrupted sleep and 24 hr of sleep deprivation. Wilcoxon signed-rank test was used to compare the distraction-induced results to the participants’ reference scores and times. Results: As compared to reference score (328), a lower performance was found for all four distractions: auditory distraction (289, p = 0.0012), fasting (302, p = 0.02), sleep interruption (304, p = 0.02) and sleep deprivation (300, p = 0.0006). In particular, PostH3 performance was influenced by all four interventions. (86 versus 50, p = 0,0012, 65, p = 0.05, 72, p = 0.05, 54, p = 0.0007 respectively). Conclusions: Virtual vitreoretinal surgery is an important tool for practicing complex surgical skills without compromising patient safety. In this study, deleterious effects on surgical performance were induced by four independent distracting factors. This knowledge is useful to optimize surgeons’ work conditions and ensuring the best possible treatment of patients.