Regular physical activity and physical fitness are closely related to a positive health status in humans. In this context, the muscle becomes more important due to its function as an endocrine organ. Muscle tissue secretes "myokines" in response to physical activity and it is speculated that these myokines are involved in physical activity induced positive health effects. Recently, the newly discovered myokine Irisin thought to be secreted by the muscle in response to physical activity and might be related to the health inducing effect by inducing browning of white adipose tissue. Speculating that myokines at least partly mediate exercise related health effects one would assume that regular physical activity and physical fitness are associated with resting Irisin concentrations in healthy humans. To investigate the association between resting Irisin concentration and either short-term physical activity, habitual physical activity, or physical fitness, data of 300 healthy participants from the cross-sectional KarMeN-study were analyzed. By applying different activity measurements we determined short-term and habitual physical activity, as well as physical fitness. Fasting serum samples were collected to determine resting Irisin concentrations by Enzyme-linked Immunosorbent Assay. Multivariate linear regression analysis served to investigate associations of the individual physical activity parameters with Irisin concentrations. Therefore, lean body mass and total fat mass (both determined by dual-energy X-ray absorptiometry) as well as age and parameters of glucose metabolism were included as confounders in multivariate linear regression analysis. Results showed that Irisin serum concentrations were not related to measures of physical activity and physical fitness in healthy humans under resting conditions, irrespective of the applied methods. Therefore we assume that if physical activity related effects are partly induced by myokines, permanently increased Irisin serum concentration may not be necessary to induce health-related exercise effects.