This study was aimed at investigating the effects of sperm centrifugation on nitric oxide (NO) and reactive oxygen species (ROS) generation as well as sperm motility and viability. Human spermatozoa were centrifuged for 10 and 30 minutes (400 x g) in the presence or absence of the NOS inhibitor, N G -nitro-L-arginine methyl ester (L-NAME); ROS scavenger, N-(2-mercaptopropionyl)Glycine (MPG) or the combination of L-NAME + MPG. Total sperm motility was significantly decreased with 30 minutes of centrifugation whereas progressive motility and cell viability were significantly decreased with 10 and 30 minutes of centrifugation. These effects were reversed with the administration of MPG or L-NAME + MPG. Ten minutes centrifugation significantly elevated ROS and NO production (P < 0.05). Thirty minutes centrifugation elevated ROS generation (P < 0.01) whereas NO was attenuated. This study has demonstrated that 10 and 30 minutes of sperm centrifugation were detrimental to both sperm motility and viability, but generally 30 minutes centrifugation was more detrimental to sperm than 10 minutes. It has also demonstrated that 10 minutes centrifugation led to both NO and ROS elevation whereas 30 minutes centrifugation led to ROS elevation and NO attenuation. We therefore recommend that sperm separation techniques should avoid using centrifugation or prolonged centrifugation in assisted reproductive technologies.