What is the central question of this study? The prevalence of sedentary behaviour in the workplace and increased daily sitting time have been associated with the development of cardiovascular disease; however, studies investigating the impact of sitting on vascular function remain limited. What is the main finding and its importance? We demonstrate that there is a marked vulnerability of the vasculature in the lower and upper limbs to prolonged sitting and highlight the importance of physical activity in restoring vascular function in a limb-specific manner. Sedentary behaviour in the workplace and increased daily sitting time are on the rise; however, studies investigating the impact of sitting on vascular function remain limited. Herein, we hypothesized that 6 h of uninterrupted sitting would impair limb micro- and macrovascular dilator function and that this impairment could be improved with a bout of walking. Resting blood flow, reactive hyperaemia to 5 min cuff occlusion (microvascular reactivity) and associated flow-mediated dilatation (FMD; macrovascular reactivity) were assessed in popliteal and brachial arteries of young men at baseline (Pre Sit) and after 6 h of uninterrupted sitting (Post Sit). Measures were then repeated after a 10 min walk (~1000 steps). Sitting resulted in a marked reduction of resting popliteal artery mean blood flow and mean shear rate (6 h mean shear rate, -52 ± 8 s(-1) versus Pre Sit, P < 0.05). Interestingly, reductions were also found in the brachial artery (6 h mean shear rate, -169 ± 41 s(-1) versus Pre Sit, P < 0.05). Likewise, after 6 h of sitting, cuff-induced reactive hyperaemia was reduced in both the lower leg (-43 ± 7% versus Pre Sit, P < 0.05) and forearm (-31 ± 11% versus Pre Sit, P < 0.05). In contrast, popliteal but not brachial artery FMD was blunted with sitting. Notably, lower leg reactive hyperaemia and FMD were restored after walking. Collectively, these data suggest that prolonged sitting markedly reduces lower leg micro- and macrovascular dilator function, but these impairments can be fully normalized with a short bout of walking. In contrast, upper arm microvascular reactivity is selectively impaired with prolonged sitting, and walking does not influence this effect.