Exposure to uninterrupted prolonged sitting leads to macro- and microvascular complications, which can contribute to increased cardiovascular disease risk. This study investigated the macrovascular and microvascular responses to 3 h of sitting that was: (i) uninterrupted (CON); and (ii) interrupted every 20 min with 1 min light intensity half squats plus calf raises (EX). Twenty healthy participants (21 [SD: 2] years; 21.5 [SD: 1.6] kg/m) were recruited to participate in this randomized cross-over trial. Macrovascular function was quantified using brachial-ankle pulse wave velocity (baPWV) and the lower- and upper-limb arterial stiffness index (ASI). Microvascular function was quantified as the medial gastrocnemius tissue oxygen saturation (StO) area under the curve (AUC) during reactive hyperemia. The baPWV did not significantly change with time ( = 0.594) or by condition ( = 0.772). The arm ASI increased by 3.6 (95% CI: 0.7 to 6.6, effect size [ES] = 0.27) with a nonsignificant condition effect ( = 0.219). There was a significant interaction effect for leg ASI ( = < 0.001), with ASI increasing (impairment) by 18.7 (95% CI: 12.1 to 25.3, ES = 0.63) for CON and decreasing (improvement) by -11.9 (95% CI: -18.5 to -5.3, ES = 0.40) for EX compared to presitting. Similarly, the AUC decreased (detrimental) by 18% (Δ = -321, 95% CI: -543 to -100, ES = 0.32) for CON and increased by 32% (Δ = 588, 95% CI: 366 to 809, ES = 0.59) for EX. The leg ASI was inversely associated with StO AUC (interclass correlation coefficient: -0.66, 95% CI: -0.51 to -0.77). These preliminary findings suggest that regularly interrupting prolonged sitting with simple bodyweight exercises may help to preserve lower-limb vascular function.