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 57±12 yrs), completed two laboratory-based conditions: five hours uninterrupted sitting (SIT) and; five hours sitting interrupted every 30 minutes by three minutes of simple resistance activities (SRA). Femoral artery function (flow mediated dilation; FMD), blood flow and shear rate were measured at zero hour, 30 minutes, one, two and five hours. Brachial FMD was assessed at zero and five hours. 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 five hours in the SIT condition, relative to SRA (p<0.001). Plasma ET-1 total AUC over five hours increased in the SIT condition compared to 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.

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