Insulin resistance in skeletal muscle is an early event in the development of diabetes with obesity being one major contributing factor. In vitro, conditioned medium (CM) from differentiated human adipocytes impairs insulin signaling in skeletal muscle cells but it is not known if insulin resistance is reversible and which mechanisms underlie this process. CM induced insulin resistance in human myotubes at the level of insulin-stimulated Akt and GSK3 phosphorylation. In Insulin-resistant skeletal muscle cells exhibit enhanced production of reactive oxygen species and ceramide as well as a downregulation of myogenic transcription factors such as myogenin and myoD. However, insulin resistance was not paralleled by increased apopotosis. Regeneration of myotubes for 24 or 48 h after induction of insulin resistance restored normal insulin signaling. However, the expression level of myogenin could not be reestablished. In addition to decreasing myogenin expression, CM also decreased the release of IL-6 and IL-8, and increased monocyte chemotactic protein-1 (MCP-1) secretion from skeletal muscle cells. While regeneration of myotubes reestablished normal secretion of IL-6 the release of IL-8 and MCP-1 remained impaired after withdrawal of CM. In conclusion, our data show that insulin resistance in myotubes is only partially reversible. While some characteristic features of insulin resistant myotubes normalize in parallel to insulin signaling after withdrawal of CM, others such as IL-8 and MCP-1 secretion and myogenin expression remain impaired over a longer period. Thus, we propose that the induction of insulin resistance may cause irreversible changes of protein expression and secretion in skeletal muscle cells.