Introduction: During night shifts, surgeons experience prolonged periods without sleep, which might impact their laparoscopic performance. Therefore, this study aimed to elucidate the effects of sleep deprivation on tissue handling and motion parameters during laparoscopic surgery. Methods: A total of 55 medical students and 20 surgeons participated in this single-center, prospective, randomized crossover trial. All students underwent standardized laparoscopic training until they reached proficiency. Afterward, all participants performed three different laparoscopic tasks twice, once sleep-deprived in the middle of the night and once well rested. Endpoints were tissue handling, defined by force exertion, instrument motion, task time, and error rate. Results: Sleep-deprived students and surgeons demonstrated a significantly lower mean force exertion but only in one task, respectively. Sleep-deprived students showed a significant slower speed of the non-dominant hand in all tasks. Surgeons were significantly slower with their dominant hand in all tasks and produced a significant shorter path length and smaller motion volume. In two of the tasks, the task completion time of sleep-deprived surgeons was significantly higher compared to the control. There was no difference regarding the occurrence of errors in both cohorts. In subgroup analysis comparing tired vs. less tired participants, both very tired students and surgeons showed a tendency, in part significant, toward higher force exertion. Conclusions: Prolonged periods of sleep deprivation appear to have an overall effect on laparoscopic skills regarding instrument motion. There is also an alteration of tissue-handling skills in terms of force exertion among very tired students and surgeons.