Exercise provides many health benefits, including improved metabolism, cardiovascular health, and cognition. We have shown previously that FNDC5, a type I transmembrane protein, and its circulating form, irisin, convey some of these benefits in mice. However, recent reports questioned the existence of circulating human irisin both because human FNDC5 has a non-canonical ATA translation start and because of claims that many human irisin antibodies used in commercial ELISA kits lack required specificity. In this paper we have identified and quantitated human irisin in plasma using mass spectrometry with control peptides enriched with heavy stable isotopes as internal standards. This precise state-of-the-art method shows that human irisin is mainly translated from its non-canonical start codon and circulates at ∼3.6 ng/ml in sedentary individuals; this level is increased to ∼4.3 ng/ml in individuals undergoing aerobic interval training. These data unequivocally demonstrate that human irisin exists, circulates, and is regulated by exercise. Display omitted •Detection and quantitation of human plasma irisin by quantitative mass spectrometry•Irisin is mainly translated from its non-canonical start codon•Irisin circulates at ∼3.6 ng/ml in sedentary individuals•Irisin levels significantly increase in individuals undergoing aerobic training Irisin is an exercise-induced myokine with beneficial metabolic functions. Its detection in human plasma has, however, been problematic. Here, Jedrychowski et al. use state-of-the-art quantitative mass spectrometry to precisely detect and quantify circulating irisin and show that it goes up in individuals undergoing aerobic interval training.