A study was conducted to evaluate the effect of sperm capacitation treatments on hamster egg penetration test (HEPT) values when using freshly obtained and zona-free frozen hamster oocytes. Semen was collected from 12 healthy donors with proven fertility and each sample processed by a sperm swim-up method, incubation in human follicular fluid (hFF), Percoll centrifugation, and refrigeration in test-yolk buffer (TYB). Capacitated sperm were then incubated with fresh and frozen-thawed eggs and the percent penetration scores and indices were calculated. Motility values of pre- and postprocessed sperm were also determined. A significant overall reduction in percent penetration scores but not penetration indices was observed when using frozen oocytes. However, within sperm treatments, differences between fresh and frozen oocytes were observed for the sperm swim-up, hFF incubation, and Percoll centrifugation groups, but not for TYB. Differences in sperm movement parameters according to sperm treatment did not appear to reflect specific penetration scores. In conclusion, despite the practical advantage of using frozen oocytes over freshly obtained oocytes for the HEPT, the specific sperm capacitation treatment must be considered a factor when the decision to use fresh or frozen oocytes is made. Furthermore, these findings reinforce the need to carefully reevaluate the predictive value of the HEPT any time modifications to the test are introduced.