Cell- and tissue culture methodology form an important base for biological, biochemical and biomedical research. Most cell culture techniques require the use of animal sera for the successful propagation of cells. However, the varying composition between batches has fuelled the need for alternatives. In the case of serum derived from animal foetuses, ethical concerns have also been raised. Here we compare the use of a platelet derived lysate (Plysate), which is currently under development as a serum substitute, in the culturing of primary human muscle cells to foetal bovine serum (FBS). In cells cultured with Plysate, differentiation into myotubes, glucose-uptake, phosphatidylinositol 3-kinase (PI3K) activity and expression and phosphorylation of ERK1/2 MAPK and PKB/Akt was impaired. Thus for primary human skeletal muscle Plysate is a sub-optimal substitute for FBS.