In this study, stress in relation to slaughter was investigated in a model system by the use of 13C, 1H, and 31P nuclear magnetic resonance (NMR) spectroscopy for elucidating changes in the metabolites in C2C12 myotubes exposed to H2O2-induced stress. Oxidative stress resulted in lower levels of several metabolites, mainly amino acids; however, higher levels of alanine were apparent in the 13C spectra after incubation with [13C1]glucose. In the 13C spectra [13C3]lactate tended to increase after exposure to increasing concentrations of H2O2; conversely, a tendency to lower levels of the unlabeled (12C) lactate were identified in the 1H spectra after stress exposure. These data indicate an increase in de novo synthesis of alanine, concomitant with a release of lactate from the myotubes to the medium at oxidative stress conditions. The changes in the metabolite levels could possibly be useful as markers for meat quality traits.