This study aimed to analyze the acute impact of exercise on serum irisin levels in 22 young (YA, 24.6 ± 3.5 yrs) and in 12 middle-aged male adults (MA, 54.6 ± 5.7 yrs) 15 min and 24 h after an incremental cycling exercise test to exhaustion. ELISA assay was used for serum irisin detection. Circulating irisin increased significantly from baseline (9.0 ± 2.0 ng/ml) to 15 min post-exercise (10.2 ± 2.0 ng/ml, P < 0.001), but the greatest increment was detected after 24 h (13.5 ± 2.5 ng/ml, P < 0.001) reaching more than 50% of the basal release. Levels were significantly higher in YA (9.7 ± 1.7 to 11.1 ± 1.8 to 14.5 ± 2.2 ng/ml) than MA (7.6 ± 1.6 to 8.7 ± 1.5 to 11.8± 2.2 ng/ml) for all measured time-points (P < 0.05). Nevertheless, MA showed a comparable increase in serum irisin levels when compared to YA. These findings highlight the importance of acute physical exercise as a countermeasure against age-related deterioration of skeletal muscle mass and function in both YA and MA.