Purpose: This systematic review and meta-analysis aims to investigate the cognitive benefits of breaking up prolonged sitting by acute physical exercises. Methods: We developed a search protocol based on the Preferred Reporting Items for Systematic Reviews and Meta-Analysis checklist (PROSPERO, CRD42021224949). A systematic literature search was performed in six electronic databases (PubMed, Scopus, Web of Science, PsycINFO, SPORTDiscus, and Cochrane Library) to identify randomized controlled trials with a within-subjects cross-over and a pre-posttest design that examined the effects of physical exercise breaks during 3–5 h of prolonged sitting on cognitive performance (e.g., executive function, attention, and memory function). Additionally, study quality was rated using the Physiotherapy Evidence Database scale. Results: Thirteen randomized controlled trials with a total of 295 participants (171 female and 124 male) were included in this systematic review. Of these studies, nine were included in our meta-analysis. The results indicated that during prolonged sitting, acute physical exercise breaks did not affect overall cognitive performance, with small between-study heterogeneity (I<25%). This is further supported by the subgroup analyses showing no differences in effect sizes between cognitive domains and different exercise intensities. Conclusions: Our findings suggest that the current evidence on interrupting prolonged sitting by acute physical exercise breaks is not univocal and that some heterogeneity exists concerning the exercise protocols exists (e.g., exercise intensity, frequency of the acute physical exercise breaks). Thus, future studies are needed to investigate whether the effectiveness of acute physical exercise breaks on cognitive performance might change as a function of different mediators (e.g., exercise characteristics, age, sleep patterns). In summary, acute physical exercise breaks during prolonged sitting allow the integration of regular physical activity in daily routines (i.e., through acute physical exercise breaks) without compromising the performance of cognitively demanding tasks.