Background: Athletes regularly compete in temperatures ranging from well below 0° Celsius (C) to those exceeding human body temperature (37°C). The lack of current understanding regarding the individual and interactive influences of exercise during hot and cold exposure is currently limiting optimization of training, racing and fueling strategies implemented by endurance athletes when performing in these extreme environments. As such, determining the degree to which ambient temperature affects substrate metabolism and performance both during and after endurance exercise in trained athletes is warranted. In the present study, we systematically investigated the environmental impact of temperature on substrate mobilization within subcutaneous abdominal adipose tissue (SCAAT) in situ and whole-body substrate oxidation during submaximal steady state cycling. Purpose: To investigate the effect of hot, cold, and neutral environmental temperatures on lipolysis within SCAAT and whole-body substrate oxidation during steady state exercise and subsequent time trial (TT) performance in endurance-trained cyclists. Methods: Ten healthy, endurance-trained male cyclists (age = 22.80 ± 2.76 yr; height = 178.58 ± 5.65 cm; mass = 74.02 ± 10.95 kg; body fat = 18.35 ± 3.37%; peak oxygen consumption (VO2peak) = 60.60 ± 4.67 ml·kg-1·min-1; power output at lactate threshold (LT) = 234.00 ± 35.00 W) participated in randomized, crossover design study that consisted of baseline testing to determine LT and VO2peak, two familiarization trials, and three experimental trials. The three experimental trials consisted of cycling in hot (39.03±1.06°C; 40.19±2.18%RH), cold (3.06±1.78°C; 41.63±5.60%RH) and thermoneutral (19.43±0.98°C; 38.97±2.23%RH) temperatures in a randomized order. The exercise protocol consisted of 25 min of cycling at 70% LT, followed immediately by 25 min at 90%LT, a 15-min break and then a 20-km TT in a thermoneutral room. In situ lipolysis of the SCAAT was measured throughout the trial with microdialysis, a minimally invasive method used to monitor SCAAT interstitial glycerol concentrations. Whole-body carbohydrate and fat metabolism was measured via indirect calorimetry, fingerstick and venous blood sampling. Results: Nine out of ten subjects were unable to complete the hot condition steady state portion of the protocol, so no TT was performed on the hot visit. No significant differences (p ≤ 0.05) were observed between groups for any measured variable aside from increased heart rate (HR) in the heat (141.16 ± 2.40 bpm) as compared to cold (124.48 ± 2.40 bpm; p < 0.001) and neutral (127.98 ± 2.40 bpm; p = 0.002) conditions. A significant time effect was observed for HR, core temperature, interstitial glycerol, blood lactate, carbohydrate and fat oxidation, VO2 and RER. No differences were observed between cold and neutral TT performances. Conclusion: SCAAT lipolysis increases during steady state exercise; however, the environmental conditions in which exercise was performed did not significantly alter SCAAT metabolism or blood flow, nor did temperature influence whole-body substrate metabolism in well-trained male cyclists. Subjects were unable to complete the TT in the heat, and no differences were observed between cold and neutral TT performance.