The aim of the present study was to evaluate the effects of various physical interventions on the function of epididymal rat spermatozoa and determine whether there are correlations among these functional parameters. Epididymal rat spermatozoa were subjected to various mechanical (pipetting, centrifugation and Percoll gradient separation) and anisotonic conditions, and sperm motility, plasma membrane integrity (PMI), mitochondrial membrane potential (MMP) and intracellular reactive oxygen species (ROS) were evaluated. Repeated pipetting caused a loss in motility, PMI and MMP (P<0.05). Minimal centrifugation force (200 g) had no effect on motility, PMI and MMP, whereas an increase in the centrifugation force to 400 g or 600 g decreased sperm function (P<0.005). Percoll gradient separation increased total motility, PMI and MMP (P<0.05). However, the spermatozoa that were subjected to mechanical interventions showed high susceptibility to a ROS stimulant (P<0.005). Anisotonic conditions decreased motility, PMI and MMP, and hypotonic conditions in particular increased basal ROS (P<0.05). In correlation tests, there were strong positive correlations among total motility, PMI and MMP, whereas ROS showed no or negatively weak correlations with the other parameters. In conclusion, the physical interventions may act as important variables, affecting functional parameters of epididymal rat spermatozoa. Therefore, careful consideration and proper protocols for handling of rat spermatozoa and osmotic conditions are required to achieve reliable results and minimise damage.