Prolonged sitting contributes to cardiovascular disease (CVD) risk. The underlying mechanisms are unknown but may include changes in arterial function and vasoactive mediators. We examined the effects of prolonged unbroken sitting, relative to regular active interruptions to sitting time, on arterial function in adults at increased CVD risk. In a randomized crossover trial, 19 sedentary overweight/obese adults (mean ± SD age 57 ± 12 yr) completed 2 laboratory-based conditions: 5 h uninterrupted sitting (SIT) and 5 h sitting interrupted every 30 min by 3 min of simple resistance activities (SRA). Femoral artery function [flow-mediated dilation (FMD)], blood flow, and shear rate were measured at 0 h, 30 min, 1 h, 2 h, and 5 h. Brachial FMD was assessed at 0 and 5 h. Plasma was collected hourly for measurement of endothelin-1 (ET-1), nitrates/nitrites, vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1). There was a significant decline in femoral artery FMD, averaged over 5 h in the SIT condition, relative to SRA ( P < 0.001). Plasma ET-1 total area under the curve over 5 h increased in the SIT condition compared with SRA ( P = 0.006). There was no significant difference between conditions in femoral or brachial shear rate, brachial FMD, nitrates/nitrites, VCAM-1, or ICAM-1 ( P > 0.05 for all). Five hours of prolonged sitting, relative to regular interruptions to sitting time, impaired femoral artery vasodilator function and increased circulating ET-1 in overweight/obese adults. There is the need to build on this evidence beyond acute observations to better understand the potential longer-term vascular-related consequences of prolonged sitting. NEW & NOTEWORTHY This is the first study to examine the effect of prolonged sitting on arterial function in adults at increased cardiovascular disease risk. We have shown that 5 h of prolonged sitting, relative to regular interruptions to sitting time, impaired femoral artery vasodilator function and increased circulating endothelin-1 in overweight/obese adults. There is now the need to build on this evidence beyond acute observations to better understand the potential longer-term vascular-related consequences of prolonged sitting.