OBJECTIVE: This study aimed to determine the dose-response relationship between prolonged sitting and vascular function in healthy individuals and those with metabolic disturbances and to investigate the acute effects, on vascular function, of interventions that target interrupting prolonged sitting. DESIGN: This is a systematic review with meta-analysis. DATA SOURCES: Ovid Embase, Ovid Medline, PubMed, and CINAHL were searched from inception to 4 December 2020. ELIGIBILITY CRITERIA: Randomized crossover trials, quasi-randomized trials, and parallel group trials where vascular function (flow-mediated dilation [FMD]) was assessed before and after an acute period of sedentary behavior was used in this study. RESULTS: Prolonged sitting resulted in a significant decrease in the standardized mean change (SMC) for lower-limb FMD at the 120-min (SMC = -0.85, 95% confidence interval [CI] = -1.32 to -0.38) and 180-min (SMC = -1.18, 95% CI = -1.69 to -0.66) time points. A similar pattern was observed for lower-limb shear rate. No significant changes were observed for any outcomes in the upper limb. Subgroup analysis indicated that prolonged sitting decreased lower-limb FMD in healthy adults (SMC = -1.33, 95% CI = -1.89 to -0.78) who had higher a priori vascular endothelial function, but not in those with metabolic and vascular dysfunction (SMC = -0.51, 95% CI = -1.18 to 0.15). Interrupting sitting with active interruptions increased the standardized mean difference for FMD, relative to prolonged sitting, but it was not statistically significant (0.13, 95% CI = -0.20 to 0.45). CONCLUSIONS: Lower-limb vascular function is progressively impaired as a consequence of prolonged sitting, up to 180 min. A similar trend was not observed in upper-limb vascular function. Subgroup analysis indicated that prolonged sitting negatively affects healthy populations, a finding not observed in those with metabolic disturbances. Regularly interrupting sitting with activity may be beneficial for those with metabolic disturbances.