Sperm cryopreservation is being increasingly used as a means of preserving the fertility of patients subjected to chemotherapy and as a method of storing donor spermatozoa prior to artificial insemination or IVF/ICSI. In this clinical context, it will be important to determine the optimal technique for isolating the spermatozoa once they have been thawed. Capturing the highest quality spermatozoa with a minimum of iatrogenic damage should not only enhance the chances of successful conception but also reduce the risk of miscarriage as well as genetic /epigenetic mutations in the embryo. To address this issue, we have cryostored human semen samples using a slow freezing protocol and Quinn's Advantage™ Sperm Freezing Medium. The samples were subsequently thawed and subjected to three types of sperm isolation procedure: swim-up from semen, density gradient centrifugation (DGC), and electrophoretic separation using the Felix™ device. A comparison of sperm quality in the unprocessed semen was also undertaken before and after freezing to provide baseline data on the impact of cryopreservation on sperm biology. Cryopreservation led to the anticipated loss of sperm motility and vitality in association with an increase in lipid peroxidation and DNA damage. Following sperm selection, all three isolation techniques resulted in an increase in sperm motility and in the case of the Felix™ and swim-up procedures, an increase in sperm vitality. Otherwise there were no significant differences between sperm separation techniques with respect to ROS generation or lipid peroxidation. However, there was a major difference in terms of DNA integrity, with the Felix™ device isolating cells exhibiting significantly lower levels of DNA damage than either DGC or swim up. This technique therefore offers some advantages over alternative isolation strategies, in terms of both the quality of the gametes isolated and the time taken to achieve the isolation.