In vitro models of muscle ageing are useful for understanding mechanisms of age-related muscle loss and aiding the development of targeted therapies. To investigate mechanisms of age-related muscle loss in vitro utilizing ex vivo human serum, fasted blood samples were obtained from 4 old (72 ± 1 years) and 4 young (26 ± 3 years) men. Older individuals had elevated levels of plasma CRP, IL-6, HOMA-IR, and lower concentric peak torque and work-per-repetition compared with young participants (P < 0.05). C2C12 myotubes were serum and amino acid starved for 1-hour and conditioned with human serum (10%) for 4 or 24-hours. After 4-hours C2C12 cells were treated with 5mM leucine for 30-minutes. Muscle protein synthesis (MPS) was determined through the surface sensing of translation (SUnSET) technique and regulatory signaling pathways measured via Western Blot. Myotube diameter was significantly reduced in myotubes treated with serum from old, in comparison to young donors (84%, P < 0.001). MPS was reduced in myotubes treated with old donor serum, compared to young serum prior to leucine treatment (32%, P < 0.01). MPS and the phosphorylation of Akt, p70S6K and eEF2 were increased in myotubes treated with young serum in response to leucine treatment, with a blunted response identified in cells treated with old serum (P < 0.05). Muscle protein breakdown signaling pathways did not differ between groups. In summary, we show that myotubes conditioned with serum from older individuals had decreased myotube diameter and MPS compared with younger individuals, potentially driven by low-grade systemic inflammation.