Previously, glycerol was the only compound known to be upregulated during rapid cold hardening. To investigate the possibility that other metabolites also change, a metabolomic analysis of aqueous-soluble metabolites was performed using gas chromatography-mass spectrometry. A total of 300 metabolites were tracked, although ~50% of peaks could not be identified with confidence. Seventeen identifiable metabolites were upregulated in response to 8 hrs of rapid cold hardening, and fourteen identifiable metabolites were downregulated. Glucose and fructose levels increased while trehalose and mannose levels were reduced, ostensibly shifting the sugar pool into forms that may be easily utilized for polyol production. In addition to the expected 2-3 fold increase in glycerol, sorbitol was also found to be upregulated, although to a lesser degree than glycerol. The greatest change was found in the amino acid pool. L-alanine levels increased two-fold, followed by increases in proline, L-leucine, and glutamine proportions. Reduced amino acids were serine, valine, and beta-alanine. A holistic view of the upregulated metabolites from this analysis shows that the glycerol synthesis pathway is enhanced during rapid cold hardening since most of the metabolites from this process are upregulated. In addition, the flesh fly upregulates many metabolites involved in the alanine biosynthesis pathway. It is clear that rapid cold-hardening for S. crassipalpis neither depends exclusively on the upregulation of a single polyol nor are the physiological effects of rapid cold hardening limited to polyol production and membrane restructuring.