To determine if insulin-mediated hyperemia is partially dependent on local muscle oxygen concentration. Sprague-Dawley rats were anesthetized, and the extensor digitorum longus (EDL) was reflected onto an inverted microscope. Intravital video microscopy sequences were recorded during baseline and hyperinsulinemic euglycemia. The muscle was reflected over a glass stage insert (Experiment 1a and 1b), or over a gas exchange chamber (Experiment 2), and microvascular capillary blood flow was recorded during sequential changes (7%-12%-2%-7%) of oxygen (O2 ) concentration. Blood flow was measured by the red blood cell supply rate (SR) in number of cells per second. All animal protocols were approved by Memorial University's Institutional Animal Care Committee. In Experiment 1a, SR increased from 8.0 to 14.0 cells/s at baseline to euglycemia (p = .01), while no significant SR variation was detected after performing a sham hyperinsulinemic euglycemic clamp (Experiment 1b). In Experiment 2, SR decreased at 12% O2 and increased at 2% O2 , compared to 7% O2 , under both experimental conditions. Magnitude of SR responses to oxygen oscillations during euglycemia were not different to those at baseline at each O2 concentration (p > .9). Our results suggest that increased blood flow observed in response to insulin is eliminated if tissue oxygen microenvironment is fixed at a given oxygen concentration.