Introduction: Acute, laboratory-based bouts of prolonged sitting attenuate lower-limb arterial endothelial-dependent vasodilation. However, the impact of habitual sedentary patterns on popliteal artery endothelial health is unclear. We tested the hypothesis that greater habitual total sedentary time, more time spent in prolonged sedentary bouts, and fewer sedentary breaks would be associated with worse popliteal flow-mediated dilation (FMD) responses. Methods: This cross-sectional study used 98 healthy participants (19–77 years, 53 females) that wore an activPAL monitor on the thigh for 6.4 ± 0.8 days to objectively measure sedentary activity and completed a popliteal ultrasound assessment to determine FMD. Both relative (%baseline diameter) and absolute (mm) FMD were calculated. Using bivariate correlation and multiple regression analyses, we examined if there were relationships between sedentary outcomes and FMD while statistically controlling for any potential confounders. Results: In the multiple regression model, age ( p = 0.006, β = −0.030, 95% CI = −0.051, −0.009) and total time in sedentary bouts > 1 hour ( p = 0.031, β = −0.005, 95% CI = −0.009, −0.001) were independent predictors of relative FMD. Age (β = −0.002, 95% CI = −0.003, −0.001), mean blood flow (β = 0.013, 95% CI = 0.002, 0.024), moderate-intensity physical activity (β = 155.9E−5, 95% CI = 22.4E−5, 289.4E−5), sedentary breaks (β = 0.036, 95% CI = 0.007, 0.066), and total time spent in sedentary bouts > 1 hour (β = −25.02E−5, 95% CI = −47.67E−5, −2.378E−5) were predictors of absolute FMD (all, p < 0.047). All independent outcomes remained significant after partially controlling for all other predictor variables (all, p < 0.031). Conclusions: Habitual prolonged sedentary bouts and sedentary breaks, but not total sedentary time, were predictors of popliteal endothelial-dependent vasodilatory function. The patterns by which sedentary time is accumulated may be more important than the total sedentary time on lower-limb arterial health.