Laparoscopic surgeons are at high risk of experiencing musculoskeletal discomfort, which is considered the result of long-lasting static and awkward body postures. We primarily aimed to evaluate whether passive and active work breaks can reduce ratings of perceived discomfort among laparoscopic surgeons compared with no work breaks. We secondarily aimed to examine potential differences in performance and workload across work break conditions and requested the surgeons evaluate working with passive or active work breaks. Following a balanced, randomized cross-over design, laparoscopic surgeons performed three 90 min laparoscopic simulations without and with 2.5 min passive or active work breaks after 30 min work blocks on separate days. The simulation included the following tasks: a hot wire, peg transfer, pick-and-place, pick-and-tighten, pick-and-thread, and pull-and-stick tasks. Ratings of perceived discomfort (CR10 Borg Scale), performance per subtask, and perceived workload (NASA-TLX) were recorded, and the break interventions were evaluated (self-developed questionnaire). Statistical analyses were performed on the rating of perceived discomfort and a selection of the performance outcomes. Twenty-one participants (9F) were included, with a mean age of 36.6 years (SD 9.7) and an average experience in laparoscopies of 8.5 years (SD 5.6). Ratings of perceived musculoskeletal discomfort slightly increased over time from a mean level of 0.1 to 0.9 but did not statistically significantly differ between conditions (p = 0.439). Performance outcomes of the hot wire and peg transfer tasks did not statistically significantly differ between conditions. The overall evaluation by the participants was slightly in favor regarding the duration and content of active breaks and showed a 65% likelihood of implementing them on their own initiative in ≥90 min-lasting laparoscopic surgeries, compared with passive breaks. Both passive and active breaks did not statistically significantly influence ratings of perceived discomfort or perceived workload in a 90 min simulation of laparoscopic surgery, with an overall low mean level of perceived discomfort of 0.9 (SD 1.4). As work breaks do not lead to performance losses, rest breaks should be tested in real-life situations across a complete working shift, where perceived discomfort may differ from this laboratory situation. However, in this respect, it is crucial to investigate the acceptance and practicality of intraoperative work breaks in feasibility studies in advance of assessing their effectiveness in follow-up longitudinal trials.