Purpose To assess and compare the acute effects of high-intensity interval training (HIIT) and moderate-intensity continuous training (MICT) on central and peripheral arterial stiffness, as well as endothelial and microvascular reactivity in healthy, physically active adults. Methods Sixteen healthy young adults (8 M/8F; age: 24 ± 3 yrs) completed a randomized crossover trial consisting of two cycling exercise protocols: HIIT (4 × 4 min at 100–105% respiratory compensation point (RCP) power) and MICT (30 min at 65% RCP power). Arterial stiffness was assessed via carotid-femoral pulse wave velocity (cfPWV) and brachial-radial pulse wave velocity (brPWV). Flow-mediated slowing (FMS) and microvascular reactivity following a vascular occlusion test were used for determination of vascular reactivity. All measurements were taken within 5–15 min following exercise cessation. Results A significant time-by-condition interaction was observed for brPWV (F = 7.08, p = 0.024), revealing a significant increase following MICT whereas no significant change was observed following HIIT (MD = 0.35 m/s). There were no interactions regarding cfPWV, although a trend towards increased cfPWV following both conditions was observed (F = 3.42, p = 0.086), as well as mean arterial pressure (F = 4.03, p = 0.063). No significant changes were observed for FMS or microvascular reactivity. Conclusion These findings demonstrate a differential acute vascular response to exercise intensity. MICT elicited a significant increase in brPWV following MICT, while no change was observed following HIIT. Central arterial stiffness response does not seem to be mediated by exercise intensity. These distinct, segment-specific responses highlight the importance of assessing both central and peripheral vascular compartments when evaluating the acute impact of aerobic exercise.