Purpose: To test the hypothesis that measures of lipid metabolism and substrate utilization differ between men and women exposed to extreme exercise-heat stress. Methods: Physically active men (n=6, 30±8 yrs, 78.8±11.0 kg, body fat 19.4±9.8%, VO2max 49.9±2.9 mL -1· kg -1· min -1 ) and women (n=6, 25±3 yrs, 60.6±4.1 kg, 25.2±6.8%, 46.3±4.5mL -1· kg -1· min -1 ) completed a treadmill-based heat tolerance test (HTT) (2 hours, 3.1 mph, 2% grade in extreme environmental conditions (104°F, 40% relative humidity). Differences (pre-, post-, change, percentage change, mean difference (MD)) within and between groups were analyzed with nonparametric inferential statistical tests. Subcutaneous abdominal adipose tissue (SCAAT) interstitial glycerol concentration and adipose tissue blood flow (i.e., ethanol Output: Input (O:I)) pre-exercise, every 30 minutes (min) of exercise, and during recovery was measured with microdialysis and assay of biofluids. Circulating concentrations of non-esterified fatty acids (NEFAs), insulin, insulin-like growth factor (IGF-1), epinephrine (EPI), and norepinephrine (NE), and cortisol were measured with commercially available kits. Resting energy expenditure (REE) was measured pre- and post-exercise using a metabolic cart. Exercising substrate utilization and metabolic heat production (MHP) were collected during 30 and 90 mins of the HTT. Results: Despite no sex differences in markers associated with stress-related metabolic signaling (cortisol, EPI, NE), endocrine response (IGF-1, insulin), or systemic concentrations of NEFAs, fat oxidation (g·min -1 ) was higher in men (vs. women) at 30 min of HTT (men (M), women (W): 0.56±0.03, 0.45±0.05, p=0.005) and at 90 min of HTT (0.61±0.05, 0.48±0.12, p=0.05). SCAAT interstitial glycerol and O:I were similar across all time points between sexes (baseline glycerol ranges (mmol·L -1 ): 92.8-530.7 (W), 321.8-588.8 (M); O:I ratio ranges: 0.42-0.79 (W), 0.55-0.77 (M). However, women had significantly greater percent change in interstitial glycerol concentration from baseline (vs. M) (W vs. M: 275 ± 287.56% vs. -8.69 ± 32.41%, p=0.003). Pre- and post-exercise REE were 16.9% (MD:371.6 kcal·day -1 , p=0.003) and 15.5% (MD:343.5kcal·day -1 , p=0.050) lower in W compared to M. Mean MHP was higher in men (MD 85W). Conclusion: Fat oxidation and SCAAT lipolysis rates were higher in men vs. women after an acute bout of strenuous exercise-heat stress. Ongoing analyses are investigating sex, energy expenditure, and heat production contributions to differences between men and women observed in our study. Our findings suggest potential for sex-specific dietary recommendations during stress exposures. DoD BA200299, HRPO E02882.1a This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.