The role of skeletal muscle in locomotive function is well defined; however recent research has indicated a role as an immunogenic organ. During systemic inflammation, muscle is exposed to pro-inflammatory factors and Tumor necrosis factor alpha (TNF-α) has been implicated as a key initiator of the early inflammatory response. Studies have indicated that muscle responds rapidly to stress by the increased production of myokines and stress or Heat Shock Proteins (HSPs) and data suggest that that these proteins may be released as signalling proteins to other cells (1, 2, 3). We hypothesise that elevated levels of TNF-α initiates the production and release of myokines and HSPs from skeletal muscle. C2C12 myotubes were cultured & differentiated in vitro (4). Myotubes were treated with TNF-α in culture media (25ng/ml media) for 3 or 6 hours; culture media and cell lysates were harvested and analysed for the presence of cytokines using Luminex multiplex (20-plex) bead analysis. RNA was isolated using TRI reagent/Rneasy clean up extraction method and reverse transcribed to single strand cDNA. Gene expression was analysed using a murine cytokine qPCRarray assay. Western blotting was used to quantify levels of HSPs in the lysate and media following TNF-α exposure (10, 25 or 50ng/ml). Cell viability was assessed by trypan blue exclusion and LIVE/DEAD staining. qPCR array analysis of cell lysates showed greater than four-fold up-regulation of complement component 3, MCP-1, CCL5, CXCL1, CXCL5, CXCL9 and CXCL10. Luminex multibead analysis showed significant (ANOVA, P-value ≤ 0.05, n=6) levels of IL-6 and RANTES located in the media at 3 hours (288pg/ml ± 70.30; 7200pg/ml ± 626.64 respectively) and 6 hours (553pg/ml ± 147.63; 2122pg/ml ± 307) following TNF-α exposure. Western blot analysis, showed significant up-regulation of intracellular levels of HSP60 and HSP70, which peaked at 3 hours following treatment and significant levels of HSP60 were detected in the culture media (ANOVA, P-value ≤ 0.05, n=6). Furthermore there was no significant loss of cell viability for up to 8 hours following TNF-α exposure. C2C12 myotubes showed significant up-regulation of myokine expression in response to TNF-α treatment and increased release of myokines. These data further support the theory that muscle can release cytokines in response to stress, thus acting as an immunogenic organ. The specific release of HSP60 supports the suggestion that heat shock proteins can be released into the extracellular environment in response to stress, and we hypothesise that they may function as danger signals for the immune system(5).