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Objective: To study the relationship between the seminal sample quality of men with varicocele and sperm capacitation., Design: Cross-sectional observational study., Setting: Academic hospital., Patient(s): Seventy-six men (19 control and 57 with varicocele) were analyzed., Intervention(s): Semen samples were submitted to a discontinuous density gradient for sperm selection. Sperm capacitation was induced using a human tubal fluid medium supplemented with bovine serum albumin., Main Outcome Measure(s): After capacitation induction, the sperm were assessed by capacitation state, computer-assisted sperm motility, mitochondrial activity, membrane integrity, acrosome reaction, and intracellular oxidative stress., Result(s): The capacitation period increased sperm motility, showing an increase in the average path velocity and a decrease in the straightness compared with sperm before capacitation (paired analysis). After capacitation, the rate of capacitated sperm, motility, and mitochondrial activity showed differences between groups (control and varicocele). The varicocele group showed lower mitochondrial activity and capacitation than the control group. On the other hand, no significant differences were observed in the other variables evaluated., Conclusion(s): Varicocele men showed less viable sperm and mitochondrial activity than control men after capacitation sperm. The induction of capacitation altered motility by increasing path velocity and decreasing straightness in all of the studied groups, evidencing the occurrence of hyperactivation., (Copyright © 2023. Published by Elsevier Inc.)