ObjectiveTo develop and validate thin film micro‐outlet devices to study microvascular blood flow responses to localized changes in skeletal muscle oxygen concentration ([O2]).HypothesisOxygen mediated blood flow regulation is initiated at the capillary level through red blood cell (RBC) oxygen saturation (SO2) dependent mechanisms.Methods10 Sprague‐Dawley rats (159‐190g) were anesthetized and instrumented to maintain cardiovascular state. The right extensor digitorum longus (EDL) muscle was blunt dissected, isolated, and reflected onto a gas exchange chamber (GEC) mounted in the stage of an inverted microscope. The GEC and EDL were coupled via a composite gas permeable membrane and a gas impermeable film fabricated with laser machined micro‐outlets of various diameters (200 μm, 400 μm, 600 μm). [O2] in the EDL was dynamically manipulated by imposing four sequential 1‐ minute [O2] oscillations between 7‐12‐2‐7% while recording intravital video for capillary RBC SO2 and hemodynamic measurement.ResultsO2 oscillations imposed on capillaries directly overlying 400 μm micro‐outlets caused significant changes in capillary SO2 at 12% GEC [O2], 86.72 ± 9.58%, and 2% GEC [O2], 46.36 ± 15.45%, compared to baseline 7% GEC [O2] 69.09 ± 13.94% (p<0.0001). SO2 in capillaries outside the outlet and within a 100 μm distance had significant changes at 2% GEC [O2], 59.38 ± 21.49% compared to baseline 7% GEC [O2], 50.88 ± 18.81% (p<0.0401). SO2 in capillaries >100 μm away from the micro‐outlets were not different following the same GEC [O2] oscillations. GEC [O2] oscillations on capillaries overlying 600 μm micro‐outlets caused significant changes in capillary SO2 at 12% GEC [O2], 83.43 ± 10.18, and 2% GEC [O2], 44.52 ± 15.77%, compared to baseline 7% GEC [O2], 66.27 ± 11.83% (p<0.0001). SO2 in capillaries outside 600 μm outlets were not different following GEC [O2] oscillations. GEC [O2] oscillations on capillaries overlying 400 μm micro‐outlets caused significant changes in capillary RBC supply rate (SR) at 2% GEC [O2], 11.96 ± 9.39 cells/s, compared to baseline 7%, 10.08 ± 7.69 cells/s, and were significantly different at 2% compared to 12% GEC [O2], 9.98 ± 7.99 cells/s (p<0.0014). GEC [O2] oscillations with 600 μm micro‐ outlets caused significant changes in SR at 2% GEC [O2], 14.14 ± 10.52 cells/s compared to baseline 7% GEC [O2], 11.10 ± 9.37 cells/s, as well as a significant change at 2% GEC [O2], compared to 12% GEC [O2], 10.16 ± 8.88 cells/s (p<0.0001).ConclusionsOur composite thin‐film micro‐outlet devices were fabricated and validated to spatially confine O2 perturbations to capillaries using micro‐outlets of varying diameters. These results demonstrate that our devices can profoundly manipulate capillary SO2 and alter capillary RBC SR in vessels directly overlying the micro‐outlet without affecting capillary SO2 at distances greater than 100 μm outside the outlets. 400 μm micro‐outlets are capable of provoking significant changes in capillary SR, with larger 600 μm outlets producing a more robust response. Our novel composite thin‐film micro‐outlet devices demonstrate that regions ~400 μm in diameter must be stimulated to elicit capillary flow responses.