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Sitting time (ST) is associated with cardiovascular disease risk factors, whereas breaking ST has been reported to be beneficial for reducing cardiovascular risk.The objective of this study is to examine the effects of breaking ST on superficial femoral artery (SFA) endothelial function.1) Prolonged sitting would induce endothelial dysfunction and changes in shear forces, and 2) breaking ST with brief periods of activity would prevent attenuation in endothelial function.Twelve nonobese men (24.2 ± 4.2 yr) participated in two randomized 3-h sitting trials. In the sitting (SIT) trial, subjects were seated on a firmly cushioned chair for 3 h without moving their lower extremities. In the breaking ST trial (ACT), subjects sat similar to the SIT trial but walked on a treadmill for 5 min at 2 mph at 30 min, 1 h 30 min, and 2 h 30 min during the sitting interval. SFA flow-mediated dilation (FMD) was assessed at baseline, 1 h, 2 h, and 3 h in each trial. Statistical analyses were performed using dependent variables SFA FMD and shear rates. Significance was set at P ≤ 0.05.In the SIT trial, there was a significant decline in SFA FMD from baseline to 3 h (baseline, 4.72% ± 3.78%; 1 h, 0.52% ± 0.85%; 2 h, 1.66% ± 1.11%; 3 h, 2.2% ± 2.15; P < 0.05 by ANOVA) accompanied by a decline in mean shear rate and antegrade shear rate but no difference in shear rate (area under the curve). By two-way repeated-measures ANOVA, ACT prevented the sitting-induced decline in FMD (baseline, 4.5% ± 2.3%; 1 h, 5.04% ± 2.85%; 2 h, 5.28% ± 5.05%; 3 h, 6.9% ± 4.5%) along with no decline in shear rates.Three hours of sitting resulted in a significant impairment in shear rate and SFA FMD. When light activity breaks were introduced hourly during sitting, the decline in FMD was prevented.