Introduction:With ageing the ability to retain skeletal muscle mass and function declines (sarcopenia), with associated “anabolic resistance” being reported. However, the impact of anabolic agents on ageing muscle cell adaptation has not been studied. The aim was therefore to utilise models of cellular ageing in the absence or presence of leucine or HMB to improve myoblast fusion. The hypothesis to be challenged was that replicatively aged cells show compromised fusion vs. control but that this would be rescued by amino acid supplementation. Methods:Control and replicatively aged C2C12murine myoblasts were supplemented with 10 mM leucine or HMB. Myotube formation was assessed morphologically, biochemically (CK, LDH, Akt, mTOR, ERK, p38 signalling) and at the gene expression level at 0 h, 24 h and 96 h. All experiments were repeated 3 times in duplicate and analyses completed using one and two-way ANOVA. Results: Although control cells showed significant fusion (P < 0.05) with time, replicatively aged myoblasts did not fuse and displayed significant reductions in CK (6-fold; P < 0.05) and significant increases in LDH (3.5-fold: P < 0.05) at 96 h vs. control. Akt, mTOR and ERK signalling over 24 h were all significantly decreased (8-fold, 3-fold and 2-fold: all P < 0.05) vs. control. Significant suppression of myogenin, IGF-I, IGF-II (200-fold, 19-fold, 52-fold: all P < 0.05) and relative increases in myostatin (12-fold; P < 0.05), with no differences in amino acid transporters were observed in replicatively aged vs. control at 96 h. However, replicatively aged cells treated with leucine significantly (2-fold: P < 0.05) increased Akt signalling at 120 minutes compared to untreated controls. HMB significantly increased Akt and mTOR at 120 minutes (both 3-fold and P < 0.05) compared to untreated control. Despite these positive signalling responses to leucine and HMB supplementation, the aged cells were still incapable of fusion by all parameters assessed. Conclusion:Replicatively aged myoblasts failed to fuse basally and with supplementation. They were responsive to leucine and HMB treatment at a signalling level, however, this was without impact on fusion. Therefore, while potentially displaying “anabolic resistance” at the level of fusion, the aged cells are not resistant to protein supplementation per se, warranting further investigation.