Irisin, a proteolytic derivative of muscle integral membrane protein FNDC5 is released into the bloodstream after endurance exercise in mice and humans. The gene is regulated by PGC-1α to activate thermogenic programs in white adipose tissue. We wanted to test the hypothesis if FNDC5 and irisin are link to exercise type and duration and if these are age-dependent. Methods: Middle-aged, non-diabetic and non-athlete men performed a 1-hour low-intensity (50% VOmax) aerobic exercise (AE) with bicycle ergometer (n=17), 21-weeks of endurance exercise (EE, n=9) and a combined endurance and resistance exercise (EE+RE, n=9), and in age matched non-exercised controls (Con, n=2). Vastus lateralis biopsies and blood samples were taken at 3-hours post-exercise. Furthermore, skeletal muscle mRNA fold changes of PGC1α and FNDC5 are investigated after 1-hour and 48-hours of single resistance exercise bout (5 × 10 RM leg press) in (a) young (n=10) and (b) old (n=11) men. FNDC5 mRNA was quantified using Taqman primers by Real-Time quantitative PCR System. PGC1α and GAPDH were quantified using in-house designed primers, iQ SYBR supermix and CFX96 Real-time PCR Detection System. Relative expression levels for FNDC5 and PGC-1α were calculated with the deltaCt method and normalized to the expression of GAPDH. Serum irisin levels were measured from serum samples using a commercial EIA kit. Results: In non-diabetic and non-athlete middle-aged men no significant changes were observed in skeletal muscle PGC-1α, FNDC5 and serum irisin after 1-hour low-intensity aerobic exercise, 21-week endurance training or 21-week endurance training combined with resistance exercise (RE) training, whereas a single RE bout increased significantly FNDC5 mRNA by 1.4-fold post-RE in young, but not in old previously untrained men. Moreover, while the increase in PGC-1α mRNA at 1-hour post-RE in old men was significant, no significant changes in FNDC5 were detected. Apart from the single RE-bout, large intra and inter-individual variations in PGC-1α, FNDC5 and serum irisin in response to endurance exercise and RE training were observed. Importantly, changes in skeletal muscle PGC-1α gene expression were not consistently accompanied by corresponding changes in FNDC5. Our findings indicate that 1) for the most part, exercise does not increase FNDC5 expression in skeletal muscle; 2) factor(s) other than PGC-1α may be involved in the regulation of FNDC5 expression. In conclusion, the large intra and inter-individual variation in FNDC5 and irisin in response to different types of exercise indicate that exercise does not seem to increase FNDC5 in skeletal muscle or circulating irisin levels. However, the increase in a subset of young individuals in acute RE warrants further studies to determine whether the effects of exercise on FNDC5 and irisin are collection time, exercise type, protocol, intensity and age-dependent.