It is well established that exercise protect againsts chronic disorders such as cardiovascular diseases (CVD), type 2 diabetes (DM2), dementia and depression. It is unclear how contracting skeletal muscles mediate metabolic positive effects on health. One of the possible explanations for the health benefit of exercise can be that regular muscle contractions mediate important messengers; myokines. Experiments have shown an increased IL-6 concentration in plasma following skeletal muscle contraction. IL-6 is being released from skeletal muscle following a meal. This suggest that myokine release can also be modulated with diet. In the present study cultured human myotubes (an in vitro model for skeletal muscle) were incubated in serum-free medium with and without energy-carrying metabolites for different periods of time. The purpose was to investigate the release of peptides and proteins by cultured human myotubes and further study the effect of energy-carrying metabolites on the release. The release of proteins was analyzed by the use of ELISAs and proteomic analysis performed on conditioned media from the cultured human myotubes. The inhibitor Brefeldin A was used to explore whether the release of proteins was due to secretion or leakage. The results show that cultured human myotubes release the proteins interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-8 (IL-8) and interleukin-13 (IL-13), and that the release perhaps can be influenced by different energy-carrying metabolites. The mean concentration of the different proteins measured in the media was generally lower when the myotubes were incubated in media containing the inhibitor Brefeldin A (BFA), indicating that the release of proteins from myotubes is due to secretion. Our studies support the theory that myotubes release proteins with hormonal functions, and that the release might be influenced by energy-carrying metabolites.