BACKGROUND: Increasing evidence highlights that accumulating sitting time in prolonged bouts is detrimental to cardiometabolic health. OBJECTIVES: This systematic review aimed to compare the effects of fractionating prolonged sitting with frequent short bouts of standing and light-intensity walking on cardiometabolic health markers and conduct a meta-analysis for differences in systolic blood pressure (SBP), postprandial glucose and insulin. METHODS: Experimental randomised crossover trials with at least three intervention arms that assessed interrupting sitting with frequent short bouts of standing and light-intensity walking over a single day compared to a prolonged sitting condition were retrieved. These studies measured at minimum one marker of cardiometabolic health in adults > 18 years. An electronic search was completed on the 2nd of August 2021, searching PubMed and Web of Science Core Collection, Scopus, Embase, Cochrane Library and APA PsycINFO. Risk of bias was assessed using a modified Downs and Black checklist. A meta-analysis was conducted using calculated Cohen's d quantifying the magnitude of difference between experimental conditions. RESULTS: Seven studies met the inclusion criteria for the systematic review. All seven studies were included within the meta-analysis for postprandial glucose, four studies were pooled for postprandial insulin and three for SBP. Biomarkers of cardiometabolic health were discussed qualitatively if fewer than three studies measured and reported the variable. A meta-analysis of seven acute, 1-day randomised crossover trials that sampled mixed-sex adults (aged > 18 years) who were predominately overweight or participants with obesity found that standing as an interruption to prolonged sitting significantly reduced postprandial glucose (∆ = - 0.31, 95% CI - 0.60, - 0.03; z = - 2.15, p < 0.04) but had no significant effect on insulin or SBP. Light-intensity walking was shown to significantly attenuate postprandial glucose (∆ = - 0.72, 95% CI - 1.03, - 0.41; z = - 4.57, p < 0.001) and insulin (∆ = - 0.83, 95% CI - 1.18, - 0.48; z = - 4.66, p < 0.001) compared to continued sitting. When comparing light-intensity walking breaks compared to standing breaks a significant reduction in glucose (∆ = - 0.30, 95% CI - 0.52, - 0.08; z = -2.64, p < 0.009) and insulin (∆ = - 0.54, 95% CI - 0.75, - 0.33; z = -4.98, p < 0.001) was observed. Both standing and light-intensity walking showed no effect on SBP. CONCLUSIONS: Frequent short interruptions of standing significantly attenuated postprandial glucose compared to prolonged sitting; however, light-intensity walking was found to represent a superior physical activity break. The feasibility and longitudinal implications of breaking sedentary behaviour with light-intensity walking should be investigated in a free-living setting. REGISTRATION: Not available.