Background and objective: The effect of exercise on the plasma concentration of high-molecular weight (HMW) adiponectin (i.e. the biologically active form of circulating adiponectin) and the possible role of HMW adiponectin in mediating the exercise-induced enhancement of insulin action are not known. The aim of this study was to evaluate the relationship between the post-exercise increase in insulin sensitivity and plasma HMW adiponectin concentration. Design and methods: We measured total and HMW adiponectin concentrations in plasma using an ELISA kit, and insulin sensitivity using the updated homeostasis model assessment of insulin sensitivity (HOMA2-IS) score in the basal, overnight fasted state, once ∼12 h after a single bout of moderate-intensity endurance exercise and once after an equivalent period of rest, in 27 healthy men and women (age: 29±1 years and body mass index: 24.7±0.8 kg/m2). Results: The HOMA2-IS score was 18±7% greater after exercise than after rest (229±20 and 196±17 respectively; P=0.006), whereas the concentrations of total adiponectin (7.8±0.5 and 7.7±0.5 mg/l respectively; P=0.597) and HMW adiponectin (3.0±0.3 and 3.0±0.3 mg/l respectively; P=0.625) were not different. The exercise-induced change in HOMA2-IS score was not related to changes in total and HMW adiponectin concentrations (P>0.3). Conclusions: Changes in HMW adiponectin concentration are not involved in the acute exercise-induced enhancement of insulin action.