The energy restriction is the most common nutritional approach to treat obesity, whose efficiency depends on oxidative response against changes in body weight. In that context, the aim of the present work was to in vivo examine the mitochondrial oxidation of obese volunteers by the 2-keto[1-(13)C]isocaproate breath test, before and after weight loss. Thirty-two volunteers (men and women) participated: 16 controls (body mass index: 19.0-27.0 kg/m2), and 16 obese (body mass index: 30.0-41.6 kg/m2) who followed a caloric restriction program for 10 weeks (-500 kcal). Before and after dieting, the 2-keto[1-(13)C]isocaproate breath test was performed by ingestion of 1 mg/kg tracer and 20 mg/kg L-leucine, dissolved in 200 ml orange juice. Breath samples were recovered at baseline and at 10 min intervals for 2 h after ingestion. The 13C-enrichment in breath was measured by isotope ratio mass spectrometry and the percentage of mitochondrial oxidation of tracer (%13C) was calculated. The percentage of oxidized tracer marginally tended to be lower in obese than in controls (25.1 +/- 5.5%, vs 27.5 +/- 4.0%, p = 0.175). After the intervention, the mean of weight loss was -7.8% +/- 3% p < 0.001), and the mitocondrial oxidation of the tracer statistically increased (25.1 +/- 5.5% vs 34.3 +/- 5.2%, p < 0.001). In fact, the body weight and the percentage of oxidized 2-keto[1-(13)C]isocaproate were inversely related (r = -0.34, p = 0.018). Thus, the 2-keto[1-(13)C]isocaproate in vivo showed the mitochondrial adaptation of obese volunteers treated by caloric-restriction intake and provided new information about the weight loss process induced by an hypocaloric diet.