Abstract  Recently contradictory findings concerning the function of irisin and its precursor gene, skeletal muscle FNDC5 in energy homeostasis and the regulatory role of exercise and PGC‐1α therein have been reported. We therefore evaluated whether muscle FNDC5 mRNA and serum irisin are exercise‐responsive and whether PGC‐1α expression is associated with FNDC5 expression. The male study subjects performed single exercises either 1‐hour low‐intensity aerobic exercise (AE) (middle‐aged, n = 17) or a heavy‐intensity resistance exercise (RE) bout (young n = 10, old n = 11), or long‐term 21‐weeks endurance exercise (EE) training alone (twice/week, middle‐aged, n = 9) or combined EE and RE training (both twice/week, middle‐aged, n = 9). Skeletal muscle mRNA expression was analyzed by quantitative PCR and serum irisin by ELISA. No significant changes were observed in skeletal muscle PGC‐1α, FNDC5 and serum irisin after AE, EE training or combined EE+RE training. However, a single RE bout increased PGC‐1α by 4‐fold in young and by 2‐fold in old men, while FNDC5 mRNA increased by 1.4‐fold post‐RE only in young men. Changes in PGC‐1α or serum irisin were not consistently accompanied with changes in FNDC5. In conclusion, for the most part, neither longer‐term nor single exercise markedly increases skeletal muscle FNDC5 expression or serum irisin. Therefore their changes in response to exercise are likely random and not general excluding the confirmation of any definitive link between exercise and FNDC5 expression and irisin release in humans. Moreover, irisin and FNDC5 did not associate with glucose tolerance and overweight or metabolic disturbances, respectively. Finally, factor(s) other than PGC‐1α and transcription may regulate FNDC5 expression. This article is protected by copyright. All rights reserved