Background: Sedentary behaviors have increased in recent years and their consequences have led the World Health Organization to make recommendations for promoting a more active lifestyle. The school environment has been defined as a key place for achieving this objective for children and adolescents. This systematic review and meta-analysis aims to analyze the effect of active-break interventions for interrupting prolonged sitting times during school-time on physical activity (PA) and sedentary behavior (SB), at school, in childhood and youth. Methods: A systematic review and meta-analysis were carried out, including clinical trials aimed at assessing the effects of interrupting prolonged sitting at school with active breaks on in-school PA and/or SB. Multimodal and static interventions were excluded. Six databases were analyzed: Medline, WOS, Cochrane Library, SPORT Discus, CINAHL and EMBASE. PA, SB; moderate-to-vigorous physical activity (MVPA) were the variables considered. Results: Nine studies were included, with a total of 2145 children between 6 and 12 years old. The heterogeneity in the duration (five-sixty min), the frequency (one-three times per-day up to three times per week), and duration (five days to three years) of the interventions was detected. The meta-analyses for in-school PA, MVPA, and SB were performed, showing a significant improvement in both PA and MVPA. Conclusions: Interrupting prolonged sitting with active-based school interventions could improve PA and MVPA levels during school time. (PROSPERO: CRD42022358933).