Prolonged sitting in a mild hypercapnic environment impairs peripheral vascular function. The effects of sitting interruptions using passive or active skeletal muscle contractions are still unclear. Therefore, we sought to examine the vascular effects of brief periods (2 min every half hour) of passive and active lower-limb movement to interrupt prolonged sitting with mild hypercapnia in adults. Fourteen healthy adults (24±2y) participated in 3 experimental visits sitting for 2.5h in a mild hypercapnic environment (CO 2 =1500 ppm): control (CON, no limb movement), passive lower-limb movement (PASS), and active lower-limb movement (ACT) during sitting. At all visits, brachial and popliteal artery flow-mediated dilation (FMD), microvascular function, plasmatic levels of nitrate/nitrate and endothelin-1, and heart rate variability were assessed before and after sitting. Brachial and popliteal artery FMDs were reduced in CON and PASS (Ptextless0.05) but were preserved (Ptextgreater0.05) in ACT. Microvascular function was blunted in CON (Ptextless0.05) but was preserved in PASS and ACT (Ptextgreater0.05). Additionally, total plasma nitrate/nitrite was preserved in ACT (Ptextgreater0.05) but was reduced in CON and PASS (Ptextless0.05), and endothelin-1 levels were decreased in ACT (Ptextless0.05). Both passive and active movement induced a greater ratio between the low frequency and high frequency band for heart rate variability (Ptextless0.05). For the first time, we found that brief periods of passive leg movement can preserve microvascular function, but that an intervention that elicits larger increases in shear rate, such as low-intensity exercise, is required to fully protect both macrovascular and microvascular function and circulating vasoactive substance balance.