Introduction: Single bouts of prolonged bent-legged sitting attenuate popliteal endothelial-dependent vasodilation (as assessed via flow-mediated dilation [FMD]), which is partially attributed to arterial ‘kinking’. However, the impact of knee-flexion angle on sitting-induced popliteal FMD is unknown. The objective of this study was to perform separate laboratory and free-living studies to test the hypotheses that: (1) popliteal FMD impairments would be graded between knee flexions at 90° (bent-legged sitting) > 45° > 0° (straight-legged sitting) following a 3-hour bout of sitting; and (2) more habitual time spent bent-legged sitting (< 45°) would be associated with lower FMD. Methods: The laboratory study included eight young, healthy adults (24 ± 2 years; four women) who underwent two sitting bouts over 2 days with one leg positioned at a knee-flexion angle of 0° or 90° and the opposite leg at 45° knee flexion. Popliteal FMD was assessed at pre- and postsitting timepoints. Results: Sitting-induced reductions in FMD were similar between all knee-flexion angles (all, p > 0.674). The free-living study included 35 young, healthy adults (23 ± 3 years; 16 women) who wore three activPAL monitors (torso, thigh, shin) to determine detailed sedentary postures. Time spent sedentary (624 ± 127 min/day), straight-legged sitting (112 ± 98 min/day), and bent-legged sitting (442 ± 106 min/day) were not related to relative FMD (5.3 ± 1.8%; all, p > 0.240). Conclusion: These findings suggest that knee-flexion angle-mediated arterial ‘kinking’ during sitting is not a major contributor toward sitting-induced popliteal endothelial-dependent vasodilatory dysfunction.