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Prolonged sitting induces endothelial dysfunction in healthy young adults, which has been demonstrated to be offset by intermittent fidgeting and standing. No information exists on the effect of sitting and endothelial dysfunction in sedentary middle-age adults, and whether common workplace counterinterventions (i.e., desk standing/desk pedaling) mitigate sitting-induced endothelial dysfunction.The objective of this study was to examine whether breaking up prolonged sitting with intermittent standing or underdesk pedaling prevents sitting-induced popliteal artery endothelial dysfunction in middle-age sedentary, overweight/obese office workers.We tested the hypothesis that sitting-induced leg endothelial dysfunction would be prevented by intermittent standing or desk pedaling.Thirteen middle-age, sedentary overweight/obese subjects (10 men, 3 women; age, 38 ± 3 yr; body mass index, 29.7 ± 2 kg·m−2) participated in three separate testing sessions in a randomized order: 1) 4 h of uninterrupted sitting, 2) 4 h of sitting interrupted with four 10-min bouts of standing, and 3) 4 h of sitting interrupted with four 10-min bouts of light-intensity desk pedaling. Doppler ultrasound–measured popliteal artery flow-mediated dilation and associated measures (e.g., shear rate, blood velocity) were measured immediately before and immediately after each intervention (sit, stand, and desk pedaling).Four hours of uninterrupted sitting induced a significant impairment in popliteal artery flow-mediated dilation (baseline: 3.1% ± 0.3%, post: 1.6% ± 0.5%; P < 0.05). Interestingly, neither intermittent standing (baseline: 3.2% ± 0.4%, post: 1.9% ± 0.5%; P < 0.05) nor intermittent desk pedaling (baseline: 3.2% ± 0.4%, post: 1.9% ± 0.4%; P < 0.05) was effective at preventing excessive sitting–induced endothelial dysfunction.Prolonged sitting–induced leg endothelial dysfunction cannot be prevented by brief intermittent bouts of standing or desk pedaling in middle-age sedentary overweight/obese adults.