Background <p>Human primary muscle cell (HPMC) lines derived from skeletal muscle biopsies are potentially powerful tools to interrogate the molecular pathways underlying fundamental muscle mechanisms. HPMCs retain their genome in culture, but many endogenous circulating factors are not present in the in vitro environment, or at concentrations that do not mirror physiological levels. To address the assumption that HPMCs are valid models of age and sex-specificity in human muscle research, we examined to what extent differentiated HPMC lines retain their source phenotype in culture.</p> Methods <p>Biopsies from the v<i>astus lateralis</i> muscle were collected from ten males aged 18–30, ten females aged 18–30 and ten males aged 60–75 recruited from a healthy population. A portion of the muscle was used for the establishment of 30 individual HMPC lines. The remaining sample was immediately snap frozen and stored for further analysis. RNA was extracted from muscle tissue samples and their corresponding, fully differentiated HMPCs and analysed using RNA Sequencing. To compare their transcriptomic signature, principal component analysis (PCA), differential expression analysis, single-cell deconvolution and pathway enrichment analysis were conducted in <i>R</i>.</p> Results <p>A comparison of the transcriptomic signature of 30 human muscle biopsies and their corresponding differentiated HPMCs indicated a near-complete lack of retention of the genes and pathways differentially regulated in vivo when compared to their in vitro equivalent, with the exception of several genes encoded on the Y-chromosome.</p> Conclusions <p>The diversity of resident cell populations in muscle tissue and the lack of sex- and age-dependent circulating factors in the cellular milieu likely contribute to these observations, which call for caution when using differentiated HPMCs as an experimental model of human muscle sex or age.</p>