In the present study, the metabolic effects of heat and anoxic stress in myotubes from the mouse cell line C2C12 were investigated by using a combination of 13C, 1H, and 31P nuclear magnetic resonance (NMR) spectroscopy and enrichment with [13C]-glucose. Both the 13C and the 1H NMR spectra showed reduced levels of the amino acids alanine, glutamate, and aspartate after heat or anoxic stress. The decreases were smallest at 42°C, larger at 45°C, and most pronounced after anoxic conditions. In addition, in both the 1H and the 31P NMR spectra, decreases in the high-energy phosphate compounds adenosine triphosphate and phosphocreatine with increasing severity of stress were identified. At anoxic conditions, an increase in 13C-labeled lactate and appearance of glycerol-3-phosphate were observed. Accumulation of lactate and glycerol-3-phosphate is in agreement with a shift to anaerobic metabolism due to inhibition of the aerobic pathway in the mitochondria. Conversely, lower levels of unlabeled (12C) lactate were apparent at increasing severity of stress, which indicate that lactate is released from the myotubes to the medium. In conclusion, the metabolites identified in the present study may be useful markers for identifying severity of stress in muscles. © 2010 Elsevier Inc. All rights reserved.