Introduction:The ability to retain skeletal muscle mass and function declines with ageing (sarcopenia). Myoblast fusion is integral to the efficient regeneration and growth of muscle. Using a relevant model, we investigated mechanisms during early differentiation involved in preventing aged myoblast fusion. The hypotheses being challenged were: 1) during early differentiation, aged cells have impairments in cellular signalling and gene expression of markers of myoblast fusion, 2) aged cells have decreased abundance and turnover of specific proteins that are associated with inhibited cell fusion. Methods:Control and replicatively aged (do not fuse but are not senescent) murine C2C12myoblasts were cultured for 96 h and myotube formation was assessed morphologically and biochemically (creatine kinase (CK)). During early myoblast differentiation (0-24 h) intracellular signalling (Akt, mTOR, ERK, p38) and gene expression (IGF-I, myogenin, ID3) were assessed. Differentiatingaged and control myoblasts were grown in the absence and presence of deuterium oxide (D20). Samples were lysed at 0 h and 24 h and analysed using LC-MS/MS to measure changes in the protein abundance and to determine fractional synthesis rate (FSR) on a protein by protein basis. Experiments were repeated 3 times in duplicate. Data were analysed using one-way ANOVA. Results: Control cells exhibited significant fusion (P < 0.05) with time, whereas aged myoblasts did not fuse and displayed significant reductions in CK (6-fold: P < 0.05) vs. control at 96 h. Akt and mTOR activation over 24 h were significantly decreased (8-fold and 3-fold respectively: both P < 0.05) vs. control. Significant suppression of myogenin, IGF-I (1000-fold and 10-fold; both P < 0.05) and a significant increase in ID3 expression (4-fold: P < 0.05) were observed at 24 h vs. control. Aged myoblasts had 33 ribosomal proteins with significantly lower (P < 0.01: FDR 10 %) abundance vs. control myoblasts. Ten metabolic enzymes with significantly greater (P < 0.01: FDR 10 %) abundance in aged myoblasts vs. control myoblasts. The average synthesis rate of proteins in aged myoblasts (0.47 ± 0.34 %/h) was significantly less (1.3-fold; P < 0.05) vs. controls (0.59 ± 0.28 %/h) and there were 157 individual proteins with relative differences in FSR. Conclusion:Replicatively aged myoblasts failed to fuse, illustrated morphologically and confirmed biochemically. Suppressed levels of Akt and mTOR activation, together with reductions in myogenin and IGF-I and increases in ID3 may underpin this process. Aged myoblasts had reduced abundance of ribosomal proteins and altered energy demands, with reduced protein turnover compared to control. Therefore, adaptations in multiple pathways during the initial 24 h of differentiation appear to underpin the inability of aged cells to fuse.