Background: Skeletal muscle stem cells are responsible for the regenerative capacity of skeletal muscle and undergo activation, proliferation, and differentiation upon injury, in order to support muscle repair. Apolipoprotein E deficient (ApoE KO) mice develop hyperlipidaemia and atherosclerosis and exhibit increased oxidative stress and triglyceride contents in skeletal muscle. Experimental evidence suggests that ApoE KO mice have impaired skeletal muscle regeneration after injury. However, the skeletal muscle stem cell function of ApoE KO mice, remains largely unknown. We hypothesised that the attenuated regenerative capacity of the ApoE KO mice derives from deficits in skeletal muscle stem cell function. Therefore, we investigated the impact of ApoE deficiency on the proliferation and differentiation profiles of muscle stem cells. Methods: Muscle stem cells from the extensor digitorum longus and biceps brachii were isolated from ApoE KO and wild type mice for single fibre culture experiments ex vivo and isolated stem cell cultures in vitro. Isolated stem cell proliferation and differentiation, morphology, immunohistochemistry and gene expression analysis were performed. Cultures were treated with 0.5mM palmitate for 24h in relevant experiments. Cell culture experiments were conducted with n=6-9 technical replicates and n=2-3 independent experiments. Significant differences were detected by the Mann-Whitney U test for p<0.05. Results: No significant differences in myogenic proliferation were observed between wild type and ApoE KO mice in single fibres after 48h in culture. However, impaired differentiation of ApoE KO mouse muscle stem cells was evident on single fibres ex vivo after 72 hours, based on expression patterns of myogenin. Furthermore, palmitate treatment resulted in loss of stemness and increased commitment to differentiation (i.e. Pax7-ve/MyoD+ve cells). Isolated muscle stem cells from ApoE KO mouse displayed impaired proliferation as well as differentiation, with morphological studies resulting in reduced myotube width and length and fusion-index, an indicator of reduced myotube fusion. These results were exacerbated by application of palmitate in both wild type and ApoE KO. These findings are strengthened by reduced gene expression for key factors regulating skeletal muscle fusion of myogenic progenitors, cell fate and contractility (i.e. MyoD, Scrib1, myogenin, Bex1, tmem8c, srf, mhc1 and acta1). Conclusion: We report for the first time impaired skeletal muscle stem cell function in hyperlipidaemic ApoE KO mice, which may account for the impaired muscle regenerative capacity of this model. These data establish a link between muscle stem biology and impaired myogenesis in an experimental model of atherosclerosis and systemic hyperlipidaemia with implications for the myopathies seen in cardiovascular disorders.