Replicative senescence of cells in culture has been used as a model to study the ageing process in human muscle (Bigot et al., 2008). However, the relevance of this approach to in vivo ageing remains unclear. The aim of this study was to investigate if in vivo muscle ageing differs from in vitro muscle ageing by studying primary human muscle cells obtained from young and elderly people, before and after they reached replicative senescence. Several parameters associated with ageing process were compared: ability to undergo differentiation; presence of DNA damage; and production of TGF-β. Muscle biopsy samples were taken under local anaesthesia from the vastus lateralis muscles of 5 young (aged, 23-25) and 4 healthy elderly (aged, 67-82) subjects. Cells were cultured in a skeletal muscle cell growth medium until they reached senescence. The proportion of muscle cells (positive for desmin expression), differentiating cells and cells with double strand DNA breaks (positive for γ-H2AX) was assessed by immunocytochemistry, whereas TGF-β production was assessed in conditioned medium using a Luminex based assay. Cells were plated in growth medium in 96 well dishes at densities of 2000 (desmin, γ-H2AX) and 7000 (myogenin, MHC, TGF-β) cells/well. Cells were fixed 24 hours after plating for assessment of desmin and γ-H2AX or had their medium replaced with a serum free medium to induce differentiation. Marked heterogeneity between the different myoblast cultures was observed during multiple passaging. Several populations of cells maintained their initial desmin content over time (50-94%) and underwent 1-8 mean population doublings. However, others lost their desmin positive cells over time in culture (50-95% starting, 0% at senescence) and underwent 15-22 MPDs. Populations that did not maintain desmin expression were discarded from further analysis after the initial characterisation. The main finding of the study is that there is no difference in any of the parameters measured between the cells obtained from old and young people that had not undergone replicative senescence. However, the senescent cells had a decrease in the expression levels of myogenin (50 ± 3% young, 49 ± 3% old and 6 ± 1% senescent, P<0.001) after three days of differentiation and MHC (71 ± 2% young, 70 ± 1.4% old and 15 ± 1% senescent, P<0.001) after five days of differentiation, higher expression of DNA damage markers γ-H2AX (7 ± 1% young, 8 ± 1% old and 90 ± 4% senescent, P<0.001) and increased TGF-β secretion (111 ± 13pg/ml young, 115 ± 19 pg/ml old and 268 ± 11pg/ml senescent, P<0.001). This questions the use of senescence in culture as a model of in vivo ageing. The findings also point to a potential link between DNA damage, TGF-β production and inhibition of differentiation that should be further investigated.