We aimed to investigate the effects of two physical exercise models, voluntary physical activity (VPA) and endurance training (ET) as preventive and therapeutic strategies, respectively, on lipid accumulation regulators and mitochondrial content in VAT of rats fed a high-fat diet (HFD). Sprague-Dawley rats (6 weeks old, n=60) were assigned into sedentary and VPA groups fed isoenergetic diets: standard (S, 35 kcal% fat) or HFD (71 kcal% fat). The VPA groups had free access to wheel running during the entire protocol. After 9 weeks, half of the sedentary animals were exercised on a treadmill while maintaining the dietary treatments. The HFD induced no changes in plasma non-esterified fatty acids (NEFA) and glycerol levels and decreased oxidative phosphorylation (OXPHOS) subunit IV and increased truncated/full-length sterol regulatory element-binding transcription factor 1c (SREBP1c) ratio in epididymal white adipose tissue (eWAT). VPA decreased plasma glycerol levels, aquaglyceroporin 7 (AQP7) and increased subunit I of cytochrome c oxidase (COX) protein, in standard diet fed animals. Eight weeks of ET decreased body weight, visceral adiposity and adipocyte size and plasma NEFA and glycerol levels, as well as AQP7 protein expression in eWAT. ET increased fatty acid translocase (FAT/CD36), mitochondrial content of complexes IV and V subunits, mitochondrial biogenesis and dynamic (mitofusins and optic atrophy 1)-related proteins. Moreover, lipogenesis-related markers (SREBP1c and acetyl CoA carboxylase) were reduced after 8 weeks of ET. In conclusion, ET-induced alterations reflect a positive effect on mitochondrial function and the overall VAT metabolism of HFD-induced obese rats.