In vitro skeletal muscle culture models provide important insight into the cellular mechanisms which underpin skeletal muscle physiology and metabolism in health and disease. The establishment of a model that can be cultured in physiological concentrations of glucose is an important factor in its translatability to more complex models and systems. Using the human skeletal muscle cell line, LHCN-M2 myoblasts, we aimed to determine the effects of different concentrations of glucose in culture media on cell viability, proliferation, ATP production and differentiation. LHCN-M2 myoblasts were cultured in NORM (1 g· L- 1) or HIGH (3.8 g· L- 1) glucose growth media, and cell viability, ATP production, and proliferation were measured. Immunofluorescence microscopy was used to determine LHCN-M2 differentiation into multinucleated myotubes with increasing concentrations of human serum (0.5%, 1% and 2% v/v). There were no differences in the viability, proliferation or basal ATP production rates of LHCN-M2 cells grown in NORM compared to HIGH glucose (P > 0.05). Morphological analysis revealed that myotube area was greater when differentiated in 2% compared to 0.5% human serum (P = 0.02), but myotube number and fusion index were unaffected (P > 0.05). These findings demonstrate that LHCN-M2 cells are capable of proliferating and differentiating into multinucleated myotubes under normal glucose concentrations in the culture media. Further work is required to determine the implications of media glucose concentration on the wider metabolic function and phenotype of LHCN-M2 myoblasts cells and myotubes.