Objective The purpose of the study was to determine the effects of passaging on retention of donor phenotypic characteristics in primary human myotubes. Methods We established primary muscle cultures and serial passaged myotubes from physically active, sedentary lean, and individuals with type 2 diabetes (T2D). We measured maximal ATP synthesis capacity (ATPmax) and resting ATP flux (ATPase) in vivo by 31P magnetic resonance spectroscopy, type-I fibers and intramyocelluar lipid (IMCL) in vastus lateralis tissue using immunohistochemistry techniques, and oxidative phosphorylation complexes (OXPHOS) by western immunoblotting. Similar in vitro measures for lipid and type-I fibers were made in myotubes, along with mitochondrial content measured by Mitotracker. Results Passage 4 and 5 measures for myotubes correlated positively with in vivo measurements for percent type-I fibers (P4: r=0.62, p=0.02; P5: r=0.69, p=0.01), ATPmax (P4: r=0.54, p=0.03; P5: r=0.47, p=0.05), and OXPHOS (P4: r=0.66, p=0.04; P5: r=0.77, p=0.006). No correlations were observed for IMCL. However, passage 4 measures for myotubes correlated with passage 5 measures for percent type-I fibers (r=0.70, p=0.01), IMCL (r=0.89, p<0.001), and mitochondrial content (r=0.51, p=0.03). Conclusions Myotubes through the first two passages following immunopurification (referred to as passage 4 and 5) reflect mitochondrial and type-I fiber content in vivo phenotype of the donor.