Background: Early abnormalities in adipose tissue dietary fatty acid storage have been proposed as an important mechanism linking obesity to the development of insulin resistance and type 2 diabetes (T2D). Although exercise has been shown to improve peripheral insulin sensitivity, the mechanisms driving these changes and the impact on fatty acid fluxes are not fully understood. Here, we investigated the effects of a single high-intensity interval training (HIIT) session and 12-weeks (3×/week) of HIIT training on postprandial metabolism in women and men (45-75 years-old; BMI > 25 kg/m2) with prediabetes. Methods: Individuals participated in three postprandial metabolic protocols: before training, 18-24 h after an exercise session and after 12 weeks of training. Participants ingested a standard liquid meal (853 kcal; 33% fat, 18% protein, 49% carbohydrates) containing U-13C palmitate and the long-chain fatty acid PET analog 18F-FTHA. Whole-body PET scans were performed 3, 4, 5 and 6 h after consuming the meal to quantify organ-specific dietary fatty acid partitioning and postprandial fatty acid metabolism. Results: Preliminary results demonstrate that there were no significant differences between the three postprandial protocols for suda and 6-h AUG of insulin (n = 9). For circulating lipids, there were no significant difference between baseline and after 12 weeks of exercise in the 6-hour AUG of plasma18F- triglycerides (TG) (-1.0 ± 17.8 %ID/100 mL·6 h),18F- chylomicron-TG (-0.1 ± 13.8% ID/100 mL·6 h) and18F-VLDL-TG (-1.3 ± 4.5 %ID/100 mL·6 h) (n = 13). Evaluation of organ-specific dietary fatty acid partitioning is currently underway. Conclusions: Our full cohort (n = 48) will allow us to better characterize postprandial responses, including the biodistribution of dietary fatty acids, following a single session and after 36 sessions of HIIT in a prediabetic population.