Background and Objectives: Skeletal muscles are considered to be the main source of circulating irisin, both at rest and during physical activity. The aim of this study was to investigate the connection between irisin, body composition, and energy metabolism in humans. Materials and Methods: Serum irisin concentrations before and after acute aerobic exercise on a treadmill in 84 healthy adults were measured and their association with body composition and energy expenditure (EE) (obtained from indirect calorimetry) was determined. Results: The total pre-exercise irisin concentrations in males and females were similar, but higher in females when expressed per body mass kg (p < 0.001). There was an association between pre-exercise irisin per body mass kg, visceral fat rating (rho = −0.52, p = 0.001), and lean tissue % (rho = 0.41, p < 0.05) in males and lean body mass index (LBMI) (rho = −0.59, p < 0.001) in females. The pre-exercise irisin concentration correlated with the resting metabolic rate (RMR) in both sexes (rho = 0.44 in males, rho = 0.36 in females; p < 0.05), but with walking, running, and the EE difference from RMR in running (Δ running EE) in males only (rho = 0.32 to 0.37, p < 0.05). There was no significant change in irisin concentration after exercise in 58% of participants, while it decreased in 23%, and increased in 19%. In male subjects with no change in irisin concentration after exercise, running (p < 0.05) and Δ running EE per body mass kg (p < 0.05) were higher than in those with decreased irisin concentration. Conclusions: These findings indicate that the association of irisin concentration with body composition and EE parameters has sex-dependent differences, and acute exercise can lead to various changes in post-exercise irisin levels.

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