Both exercise and calorie restriction interventions have been recommended for inducing weight-loss in obese states. However, there is conflicting evidence on their relative benefits for metabolic health and insulin sensitivity. This study seeks to evaluate the differential effects of the two interventions on fat mobilization, fat metabolism, and insulin sensitivity in diet-induced obese animal models. After 4 months of ad libitum high fat diet feeding, 35 male Fischer F344 rats were grouped (n = 7 per cohort) into sedentary control (CON), exercise once a day (EX1), exercise twice a day (EX2), 15% calorie restriction (CR1) and 30% calorie restriction (CR2) cohorts. Interventions were carried out over a 4-week period. We found elevated hepatic and muscle long chain acylcarnitines with both exercise and calorie restriction, and a positive association between hepatic long chain acylcarnitines and insulin sensitivity in the pooled cohort. Our result suggests that long chain acylcarnitines may not indicate incomplete fat oxidation in weight loss interventions. Calorie restriction was found to be more effective than exercise in reducing body weight. Exercise, on the other hand, was more effective in reducing adipose depots and muscle triglycerides, favorably altering muscle/liver desaturase activity and improving insulin sensitivity.