Objective: To determine the effects of supplementation of preparation media with ascorbate and α-tocopherol on subsequent sperm motility and reactive oxygen species production.Design: Prospective study to analyze postpreparation human sperm motility parameters and reactive oxygen species production following antioxidant supplementation.Setting: Andrology Laboratory, Royal Maternity Hospital, Belfast, Northern Ireland.Patient(s): Sixty patients attending the Andrology Laboratory for semen analysis.Intervention(s): Normozoospermic and asthenozoospermic semen samples (n = 10 for each control and antioxidant group) were prepared by Percoll density centrifugation (95%-47.5%) in media supplemented with ascorbate or α-tocopherol to different concentrations within physiologic levels. Controls were included that were not exposed to antioxidant.Main Outcome Measure(s): Sperm motility parameters were assessed using computer-assisted semen analysis. The generation of reactive oxygen species was determined using luminol-dependent chemiluminescence.Result(s): The production of reactive oxygen species by sperm was reduced by supplementation in vitro with ascorbate and α-tocopherol. However, progressive motility, average path velocity, curvilinear velocity, straight-line velocity, and linearity were decreased significantly, with the greatest inhibition observed with the highest concentrations of antioxidants.Conclusion(s): Supplementation of preparation media with ascorbate and α-tocopherol, either singly or in combination, is not beneficial to sperm motility.