Adaptive thermogenesis, the increase in energy expenditure in response to diet or cold exposure, shows large inter-individual differences. The objective of this study was to investigate the proteins in human muscle tissue that relate to this variation. Therefore, we studied correlations between changes in expressions of proteins and increases in energy expenditure. This, in proteomic research, innovative application of widely used statistical approaches optimized the information yield in this study. The metabolic responses to cold and overfeeding in 9 lean adult male subjects were measured in a respiration chamber in a baseline condition, during three days of cold exposure, and during three days of overfeeding. After each respiration chamber measurement a muscle biopsy was taken, from which proteins were isolated and separated using 2D gel-electrophoresis. 95 spots that were significantly changed were analyzed using MALDI-TOF/TOF mass spectrometry. Of these proteins, 52 have been identified. Remarkably, many of the identified proteins that changed in expression significantly after overfeeding and after cold exposure, are part of the glycolytic pathway. However, the identified proteins are not considered to be rate limiting. After overfeeding, the abundance of these glycolytic proteins increased. Upon cold exposure, differences in glycolytic protein concentrations related significantly to the inter-individual differences in cold-induced adaptive thermogenesis. Moreover, increased abundance of ATP synthase subunits suggested an increased ATP-production. This shows that upon cold exposure ATP utilizing processes might be involved that were not apparent in the baseline situation. The results of this study stress the importance of changes in glycolytic proteins in both cold- and overfeeding-induced adaptive thermogenesis.