The ultimate goal of surgical training is consolidated skills with a consistently high performance. However, surgical skills are heterogeneously retained and depend on a variety of factors, including the task, cognitive demands, and organization of practice. Virtual reality (VR) simulation is increasingly being used in surgical skills training, including temporal bone surgery, but there is a gap in knowledge on the retention of mastoidectomy skills after VR simulation training. To determine the retention of mastoidectomy skills after VR simulation training with distributed and massed practice and to investigate participants' cognitive load during retention procedures. A prospective 3-month follow-up study of a VR simulation trial was conducted from February 6 to September 19, 2014, at an academic teaching hospital among 36 medical students: 19 from a cohort trained with distributed practice and 17 from a cohort trained with massed practice. Participants performed 2 virtual mastoidectomies in a VR simulator a mean of 3.2 months (range, 2.4-5.0 months) after completing initial training with 12 repeated procedures. Practice blocks were spaced apart in time (distributed), or all procedures were performed in 1 day (massed). Performance of the virtual mastoidectomy as assessed by 2 masked senior otologists using a modified Welling scale, as well as cognitive load as estimated by reaction time to perform a secondary task. Among 36 participants, mastoidectomy final-product skills were largely retained at 3 months (mean change in score, 0.1 points; P = .89) regardless of practice schedule, but the group trained with massed practice took more time to complete the task. The performance of the massed practice group increased significantly from the first to the second retention procedure (mean change, 1.8 points; P = .001), reflecting that skills were less consolidated. For both groups, increases in reaction times in the secondary task (distributed practice group: mean pretraining relative reaction time, 1.42 [95% CI, 1.37-1.47]; mean end of training relative reaction time, 1.24 [95% CI, 1.16-1.32]; and mean retention relative reaction time, 1.36 [95% CI, 1.30-1.42]; massed practice group: mean pretraining relative reaction time, 1.34 [95% CI, 1.28-1.40]; mean end of training relative reaction time, 1.31 [95% CI, 1.21-1.42]; and mean retention relative reaction time, 1.39 [95% CI, 1.31-1.46]) indicated that cognitive load during the virtual procedures had returned to the pretraining level. Mastoidectomy skills acquired under time-distributed practice conditions were retained better than skills acquired under massed practice conditions. Complex psychomotor skills should be regularly reinforced to consolidate both motor and cognitive aspects. Virtual reality simulation training provides the opportunity for such repeated training and should be integrated into training curricula.