Purpose: We investigated the effects of manipulating running velocity and hypoxic exposure on vastus lateralis muscle oxygenation levels during treadmill running. Methods: Eleven trained male distance runners performed 7 randomized runs at different velocities (8, 10, 12, 14, 16, 18, and 20 km·h−1), each lasting 45 seconds on an instrumented treadmill in normoxia (fraction of inspired oxygen [FiO2] = 20.9%), moderate hypoxia (FiO2 = 16.1%), high hypoxia (FiO2 = 14.1%), and severe hypoxia (FiO2 = 13.0%). Continuous assessment of Tissue Saturation Index (TSI) in the vastus lateralis muscle was conducted using near-infrared spectroscopy. Subsequently, changes in TSI (ΔTSI) data over the final 20 seconds of each run were compared between velocities and conditions. Results: There was a significant velocity × condition interaction for ΔTSI% (P textless .001, η2p=.19), with a smaller ΔTSI% decline in normoxia compared with high hypoxia and severe hypoxia at 8 km·h−1 (g = 1.30 and 1.91, respectively), 10 km·h−1 (g = 0.75 and 1.43, respectively), and 12 km·h−1 (g = 1.47 and 1.95, respectively) (pooled values for all conditions: P textless .037). The ΔTSI% decline increased with each subsequent velocity increment from 8 km·h−1 (−9.2% [3.7%]) to 20 km·h−1 (−22.5% [4.1%]) irrespective of hypoxia severity (pooled values for all conditions: P textless .048). Conclusions: Running at slower velocities in conjunction with high and severe hypoxia reduces vastus lateralis muscle oxygenation levels. Muscle ΔTSI% proves to be a sensitive indicator, underscoring the potential use of near-infrared spectroscopy as a reference index of internal load during treadmill runs.