Background: Exposure to acute prolonged sitting can result in vascular dysfunction, particularly within the legs. This vascular dysfunction, assessed using flow-mediated dilation (FMD), is likely the consequence of decreased blood flow-induced shear stress. With mixed success, several sitting interruption strategies have been trialled to preserve vascular function. Objectives: The objectives of this meta-analysis were to (1) assess the effects of acute prolonged sitting exposure on vascular function in the upper- and lower-limb arteries, and (2) evaluate the effectiveness of sitting interruption strategies in preserving vascular function. Sub-group analyses were conducted to determine whether artery location or interruption modality explain heterogeneity. Data Sources: Electronic databases (PubMed, Web of Science, SPORTDiscus, and Google Scholar) were searched from inception to January 2020. Reference lists of eligible studies and relevant reviews were also checked. Study Selection: Inclusion criteria for objective (1) were: (i) FMD% was assessed pre- and post-sitting; (ii) studies were either randomised-controlled, randomised-crossover, or quasi-experimental trials; (iii) the sitting period was ≥ 1 h; and (iv) participants were healthy non-smoking adults (≥ 18 years), and free of vascular-acting medication and disease at the time of testing. Additional inclusion criteria for objective (2) were: (i) the interruption strategy must have been during the sitting period; (ii) there was a control (uninterrupted sitting) group/arm; and (iii) the interruption strategy must have involved the participants actively moving their lower- or upper-limbs. Appraisal and Synthesis Methods: One thousand eight hundred and two articles were identified, of which 17 (22 trials, n = 269) met inclusion criteria for objective (1). Of those 17 articles, 6 studies (9 trials, n = 127) met the inclusion criteria for objective (2). Weighted mean differences (WMD), 95% confidence intervals (95% CI), and standardised mean difference (SMD) were calculated for all trials using random-effects meta-analysis modelling. SMD was used to determine the magnitude of effect, where <0.2, 0.2, 0.5, and 0.8 was defined as trivial, small, moderate, and large respectively. Results: (1) Random-effects modelling showed uninterrupted bouts of prolonged sitting resulted in a significant decrease in FMD% (WMD = − 2.12%, 95% CI − 2.66 to − 1.59, SMD = 0.84). Subgroup analysis revealed reductions in lower- but not upper-limb FMD%. (2) Random-effects modelling showed that interrupting bouts of sitting resulted in a significantly higher FMD% compared to uninterrupted sitting (WMD = 1.91%, 95% CI 0.40 to 3.42, SMD = 0.57). Subgroup analyses failed to identify an optimum interruption strategy but revealed moderate non-significant effects for aerobic interventions (WMD = 2.17%, 95% CI − 0.34 to 4.67, SMD = 0.69) and simple resistance activities (WMD = 2.40%, 95% CI − 0.08 to 4.88, SMD = 0.55) and a trivial effect for standing interruptions (WMD = 0.24%, 95% CI − 0.90 to 1.38, SMD = 0.16). Conclusions: Exposure to acute prolonged sitting leads to significant vascular dysfunction in arteries of the lower, but not upper, limbs. The limited available data indicate that vascular dysfunction can be prevented by regularly interrupting sitting, particularly with aerobic or simple resistance activities.