Dynamic exercise results in increased muscle, and decreased skin blood flow in addition to a progressive decrease in tissue oxygenation with increased workload (Peltonen et al. 2012). Cooling of skin and skeletal muscle also reduces tissue oxygenation, in part due to an increase in peripheral vasoconstriction and a decrease in blood volume (Yanagisawa et al. 2007). It is currently unclear whether tissue oxygenation decreases further during aerobic exercise when combined with cold exposure. Using near-infrared spectroscopy (NIRS) technology, we examined the effects of whole-body cooling on muscle tissue oxygenation during aerobic exercise. Healthy male subjects (N = 11), dressed in shorts and t-shirt, completed 6 exercise trials on separate days. They exercised at 50% (walking) or 70% (running) of maximal oxygen consumption (VO2max), for 60 min on a treadmill in a climatic chamber set at 22°C (Neutral, NT), 0°C (Cold, CO), or at 0°C following a pre-cooling period inducing deep tissue cooling (Cold with deep tissue cooling, CD). NIRS optodes were positioned on the muscle belly of the vastus lateralis (VL) and the gastrocnemius (GM) muscles to determine changes in skeletal muscle oxygenated (O2Hb) and deoxygenated (HHb) myo/hemoglobin levels (ΔµM.L-1). Core and skin temperatures were also measured. Measurements were collected at baseline, 10, 30 and 60 min of exercise and analyzed by ANOVA (Mean ± SE). Skin temperature decreased by 10.0 ± 0.6 and 11.1 ± 0.5°C in CO and CD, respectively, and remained low during 60 min. Core temperature decreased in CD up to 30 min only (0.65°C ± 0.08) (p < 0.001). A clear reduction in muscle oxygenation was observed in GM and VL (70% VO2max only) during exercise (p < 0.001), which was greater at 70% vs. 50% VO2max (p ≤ 0.006). The decrease in O2Hb was greater in CO (22.26 ± 1.27 µM.L-1) and NT (23.75 ± 1.43 µM.L-1) compared to CD (17.70 ± 1.57 µM.L-1) within the GM (p = 0.023) at 10 min of exercise only. In VL, however, the decrease was greater in CD compared to other conditions (p = 0.021). The increase in HHb was lower in CD (8.77 ± 3.07 µM.L-1) compared to CO (18.92 ± 6.70 µM.L-1) and NT (18.90 ± 2.84 µM.L-1) at 10 min in GM (p = 0.019). This was not observed in VL (p ≥ 0.972). Tissue oxygenation responses differed between GM and VL. Interestingly, no differences in muscle oxygenation were observed between exercise in a neutral and a cold environment without whole-body deep tissue cooling. During the early stages of aerobic exercise, tissue deoxygenation was delayed in GM with deep tissue cooling whereas a greater decrease in O2Hb and unchanged HHb was seen in VL. These results may likely be due to a change in walking and running gait mechanics to perform the same workload relying on a larger muscle mass as muscle cooling is known to reduce mechanical efficiency, thereby modulating oxygen availability and transport.