Ageing and reduced levels of physical activity are associated with desensitisation of skeletal muscle to the anabolic effects of amino acids. In vitro studies have indicated that many properties of skeletal muscle tissue are retained in human myotubes, including metabolic alterations associated with exercise and disease. However, the interaction between ageing and physical activity on amino acid sensing and growth has not been explored in human myotubes in vitro. Muscle-derived cells were isolated from biopsies taken from eight young (Y: 23.4 ± 1.9 yr), six older (O: 72.5 ± 5.0 yr), and nine older exercise trained (OT: 71.0 ± 4.1 yr, n = 9) men, and myotube cultures were generated and investigated for growth parameters and amino acid induced changes in mTORC1 signalling and protein synthesis. Our results indicated that muscle cell fusion was similar between groups, but myotube diameter was lower in cultures derived from O individuals. Despite this, mTORC1 signalling, as indicated by immunoblots for phosphorylation of mTOR, rpS6, and 4E-BP1 increased to a similar extent in response to amino acid availability in Y, O, and OT myotubes. Furthermore, measures of protein synthesis using the SUnSET assay were increased similarly between groups after the addition of amino acids. These data suggest that skeletal muscle desensitisation to amino acids with ageing is not observed in myotubes cultured in vitro, which could be reflective of the healthy individuals tested in our study or point towards the importance of the muscle niche in the impairments in muscle metabolism in ageing.