Background: Aerobic adaptations following interval training are supposed to be mediated by increased local blood supply. However, knowledge is scarce on the detailed relationship between exercise duration and local post-exercise blood supply and oxygen availability. This study aimed to examine the effect of five different exercise durations, ranging from 30 to 240 s, on post-exercise muscle oxygenation and relative changes in hemoglobin concentration. Methods: Healthy male subjects (N = 18) performed an experimental protocol of five exercise bouts (30, 60, 90,: 120, and 240 s) at 80 % of peak oxygen uptake VO2peak in a randomized order, separated by 5-min recovery periods. To examine the influence of aerobic fitness, we compared subjects with gas exchange thresholds (GET): above 60 % VO2peak (GET60+) with subjects reaching GET below 60 % VO2peak (GET60−). VO2 and relative changes in:: concentrations of oxygenated hemoglobin, deoxygenated hemoglobin, and total hemoglobin were continuously measured with near-infrared spectroscopy of the vastus lateralis muscle. Results: Post-exercise oxygen availability and local blood supply increased significantly until the 90-s exercise duration and reached a plateau thereafter. Considering aerobic fitness, the GET60+ group reached maximum post-exercise oxygen availability earlier (60 s) than the GET60− group (90 s). Conclusions: Our results suggest that (1) 90 s has evolved as the minimum interval duration to enhance local oxygen availability and blood supply following cycling exercise at 80 % VO2peak; whereas (2) 60 s is sufficient to: trigger the same effects in subjects with GET60 + .