Background: The number of studies on gender differences in psychomotor performance and sleepiness is small and the results are contradictory. The aim of this study was to assess the changes in psychomotor performance, due to 24 h of sleep deprivation in young women and men. Participants: Eighty-nine students (49 women and 40 men) took part in the study. Participants were randomized into two groups: experimental (sleep deprived) and control (non-sleep deprived). Methods: The research was carried out using computer-based tests of the Vienna Test System (COG, DT, WAFF) and pupillography (F 2 D Fit-For-Duty). Results: There were no statistically significant effects of the main genders and groups on sleepiness measured by the pupillography. There was no deterioration in the results after deprivation among women and men in the COG test. Changes were noted in the DT and WAFF tests, and their size depended on the test. The number of false responses in psychomotor test was higher in women after sleep deprivation. Conclusion: One night of sleep deprivation may not have been a negative enough stimulus for young, healthy women and men to reveal gender differences in psychomotor tests. Low sleep levels can lead to low productivity at work and accidents due to reduced vigilance. Insufficient sleep in the long term can lead to poor health, resulting in hypertension, obesity and depression.