Heat conditioning aids in acclimatization to support health and performance, yet heat safety is an important factor. This quasi-experimental pilot study investigated differences in micro-environmental conditions and physiological outcomes in n = 5 Division I collegiate American football players over different playing surfaces during summertime in the southwest U.S. Participants performed three practice sessions on hot days (∼33°C): outdoors on artificial turf (AT) and natural grass (NG); and an indoor dome (ID). Microclimate parameters, including net radiative fluxes, and physiological markers (core temperature (T c ), skin temperature (T sk ), heart rate, and hydration) were continuously and simultaneously monitored. Microclimate conditions varied across the three environments. Outdoors, the largest differences were observed in surface temperatures between AT and NG (67.0°C and 32.8°C, respectively), resulting in higher emitted longwave radiation (infrared heat), slightly increasing air temperatures. Indoors, the lack of radiation lessened the overall heat load, yet higher humidity and lower airflow were observed. Physiologically, similar baseline T c and T sk and self-reported heat stress levels were recorded. During exercise, a significantly higher T sk value was found on the AT (higher heat load), followed by the NG (moderate) and ID (lowest). The same pattern was reflected in T c , RPE, and self-reported heat stress, even with lower solar radiation on AT. No differences between environments were reported for estimated energy expenditure, pre/post-body weight, bodyweight loss, fluid intake, or sweat rate. Small changes in microclimates affect overall heat loads and measured and perceived heat stress, which coaches can use in decision-making for session type, heat safety, and/or acclimatization goals.