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Research has revealed that reactive oxygen species (ROS) negatively affect sperm function, both in vivo and in vitro. Sperm preparation techniques for assisted reproductive technologies (ART) are potential causes for additional ROS production. This study aimed to correlate the concentration of exogenous H₂O₂ with sperm motility parameters and intracellular ROS and nitric oxide (NO) levels to reiterate the importance of minimising ROS levels in ART. Human spermatozoa from 10 donors were incubated and exposed to different exogenous H₂O₂ concentrations (0, 2.5, 7.5 and 15 μm). Subsequently, motility was determined using computer‐aided semen analysis, while ROS (2,7‐dichlorofluorescin diacetate) and NO (diaminofluorescein‐2/diacetate) were analysed using fluorescence‐activated cell sorting. Results showed that H₂O₂ did affect the sperm parameters. Exogenous H₂O₂ was detrimental to motility …