Introduction During the last years it has become increasingly clear that different tissues in the body communicate with other tissues via hormonal signals synthesized and secreted to the blood as demonstrated in the last decade for the adipose tissue.Skeletal muscle accounts for approximately 40 % of the body weight of a normal weight fit person. It is known that exercise offers protection against chronic disorders such as cardiovascular diseases, type 2 diabetes, dementia, and depression. It is unclear how contracting skeletal muscles mediate metabolic effects of benefits on health, which has been called the work stimulus, the work factor or the exercise factor. It has been shown that muscle fibres produce and release the cytokine IL-6 into the circulation during exercise. It is also possible that released myokines may explain how regular muscle activity can influence mood, performance, and cognitive function.Aims The aims of this study were to investigate the release of peptides and proteins by human myotubes in culture and further study the effect of energy-carrying metabolites on the release.Methods By analyzing the conditioned media by using proteomic analysis and ELISAs, we could measure the release of proteins from human myotubes in culture. We also used Brefeldin A (BFA) to further explore whether the release was due to secretion or due to leak because of cell damage. Results The mean concentration of IL-6 from myotubes incubated in medium without BFA was 19 and 11 times higher than the concentration derived from myotubes incubated in medium with BFA, when incubating 3 and 6 hours, respectively. IL-6, IL-8, IL-7 and IL-13 were detected by Luminex Multiplex technology.Conclusion We have during this work optimized several conditions important toconcider when using myotubes as a model for identifying novel myokines. In addition to IL-6 we detected some potentially new myokines which are; IL-7, IL-8 and IL-13. Our studies support the theory that myotubes release proteins with possible hormonal functions, and that the release perhaps can be influenced by different energy carrying metabolites.