Human serum embodies the integrated systemic response to any condition or perturbation, which may regulate muscle protein synthesis (MPS). Conditioning of medium with human serum represents a physiologically relevant method of regulating MPS in vitro. The primary purpose of the study was the development of a model using ex vivo human serum to condition medium and regulate MPS in in vitro skeletal muscle cells. Four young healthy men reported to the laboratory after an overnight fast and were fed with 0.33g(kg body mass)(-1) whey protein. Blood samples were taken before (Fasted) and 60min postprandial (Fed). Fully differentiated C2C12 skeletal muscle cells were nutrient and serum deprived for 1h and subsequently treated with medium conditioned with Fasted or Fed ex vivo human serum (20%) for 4h. The MPS was measured using the surface sensing of translation technique and activation of mTOR, P70S6K and 4EBP1 by Western blot. Fasted and fed ex vivo human serum increased MPS (P<0.05). Although a strong effect (?(2)=0.36) for increased MPS in Fed relative to Fasted was observed, this was not statistically significant (P>0.05). Activation of mTOR, P70S6K and 4EBP1 was significantly increased after treatment with Fed compared with Fasted ex vivo human serum (P<0.05). Here, we developed and optimized the conditions for culture of C2C12 skeletal muscle cells, measurement of MPS and signalling in medium conditioned by ex vivo human serum. Furthermore, the functionality of the model was demonstrated by comparison of the response to medium conditioned by Fasted and Fed ex vivo human serum.