The World Health Organization has recommended breaking up sitting time to improve cardiovascular health. However, whether isometric exercise can be effectively used as a strategy to break up sitting time remains unclear. Thus, the aim of this study was to analyze the acute effects of breaking up prolonged sitting with isometric wall squat exercise (IWSE) on vascular function and blood pressure (BP) in sedentary adults. This randomized crossover trial included 17 adults (53% male, 26±6yr, 22.4±3.6 kg/m 2 ) with high sedentary behavior (≥ 6hr/d). The participants completed 2 experimental sessions in a randomized order, both sharing a common sitting period of 180min: Breaks (2-min breaks were incorporated into the IWSE, with participants maintaining their knees at the angle determined by the incremental test, which occurred every 30min) and Control (sitting for 180min continuously). Popliteal artery flow-mediated dilation (FMD) and brachial BP were measured before and at 10 and 30min after the experimental sessions. The results did not indicate significant session vs time interaction effects on popliteal FMD and brachial BP ( P >.05). A subanalysis including only participants with popliteal FMD reduction after the Control session (n=11) revealed that Breaks enhanced popliteal FMD after 10min (1.38±6.45% vs -4.87±2.95%, P =.002) and 30min (-0.43±2.48% vs -2.11±5.22%, P =.047). Breaking up prolonged sitting with IWSE mitigates impaired vascular function resulting from prolonged sitting but has no effect on BP in sedentary adults.