Sustaining a weight loss after a lifestyle intervention is challenging. The objective of the present study was to investigate if mitochondrial function is associated with the ability to maintain a weight loss. Sixty-eight former participants in an 11-12-week lifestyle intervention were recruited into 2 groups; weight loss maintenance (WLM; body mass index (BMI): 32 +/- 1 kg/m(2)) and weight regain (WR; BMI: 43 +/- 2 kg/m(2)) based on weight loss measured at a follow-up visit (WLM: 4.8 +/- 0.4; WR: 7.6 +/- 0.8 years after lifestyle intervention). Maximal oxygen consumption rate, physical activity level, and blood and muscle samples were obtained at the follow-up experiment. Mitochondrial respiratory capacity and reactive oxygen species (ROS) production were measured. Fasting blood samples were used to calculate glucose homeostasis index. WR had impaired glucose homeostasis and decreased maximal oxygen uptake and physical activity level compared with WLM. The decreased physical activity in WR was due to a lower activity level at vigorous and moderate intensities. Mitochondrial respiratory capacity and citrate synthase (CS) activity was higher in WLM, but intrinsic mitochondrial respiratory capacity (mitochondria' respiratory capacity corrected for mitochondria' content (CS activity)) was similar. ROS production was higher in WR compared with WLM, which was accompanied by a decreased content of antioxidant proteins in WR. Intrinsic mitochondria' respiratory capacity in skeletal muscle is not associated with the ability to maintain a long-term weight loss. WLM had a higher maximal oxygen uptake, physical activity level, mitochondrial respiratory capacity and CS activity compared with WR. The reduced glucose tolerance was concurrent with increased ROS production per mitochondria in WR, and could also be associated with the lower physical activity level in this group.