The purpose of this work was to determine endothelial, microvascular, skeletal muscle oxidative capacity (SMOC) and cardiorespiratory responses to an acute bout of continuous single leg cycling (SLC) and double leg cycling (DLC). Ten recreationally active men and women volunteered to participate in this investigation and reported to the laboratory on four separate occasions. Visits 1 and 2 consisted of a DLC and SLC p̌hantomdot V_mathrmO_2mathrmpeak test, while visits 3 and 4 were the experimental visits. Participants performed 30 min of continuous DLC and SLC at 60% of their DLC ȟantomdot V_mathrmO_2mathrmpeak. Before, 1 and 2 h post-exercise, measures of vascular (i.e., flow mediated dilation (FMD), reactive hyperaemia, microvascular responsiveness) and SMOC were performed. SLC resulted in significantly greater limb specific power (83 ± 23 vs. 51 ± 12 W; P textless 0.001) and carbohydrate oxidation (151 ± 40 vs. 126 ± 30 kcal; P = 0.017) compared to DLC. There was a significant reduction in % FMD following SLC (baseline: 9.4 ± 3.2%; 1 h: 6.9 ± 3.4%; P = 0.009), while there was no change following DLC. Both SLC and DLC resulted in a significant increase in SMOC (P textless 0.001) and a significant decrease in microvascular responsiveness (P textless 0.001). In conclusion, the reduction in FMD following SLC, likely brought on by greater peripheral and oxidative stress, which are key stimuli for long-term positive adaptations, may be more beneficial at improving peripheral adaptations compared to DLC. This may be particularly advantageous for those with exercise intolerance, as SLC leads to greater peripheral stress for a similar central stress.