Cryotherapy is a commonly used therapeutic modality for skeletal muscle injuries in sports medicine. Despite the widespread use of this modality, there is little known about the biochemical effects of cryotherapy in human skeletal muscle tissue of humans. PURPOSE: To determine the effects cryotherapy has on the transcriptome and metabolome of skeletal muscle. METHODS: Using a paired design, 8 healthy male subjects (mean age 24.7 years, mean BMI 22.2) received ice-cup massage or sham cup massage over a 45 cm2 area on each thigh for 15 min. Two hours after application, bilateral biopsies were taken at a depth of 2 cm from the vastus llateralis in the center of the ice or sham area. Muscle biopsies were from each leg were then subjected to microarray or LCMS-based metabolomics analysis. Differences between groups were tested using paired t-tests (α=0.05). RESULTS: Intramuscular (IM) temperature 2 cm deep to the subcutaneous layer was predicted from regression equations of skin temperature. At the end of the 15 min application, IM temperature was reduced by 29%, and by two hours remained 13% cooler than prior to administration ofering cryotherapy. Microarray analysis revealed changes in some non-coding RNAs, but no differences were found for protein coding genes. Further analysis by qPCR showed no significant differences in so- called “cold-shock” genes which have been reported to be induced in animals tissues exposed to substantial cooling. Metabolomics analysis of over 60 metabolites involved in glycolysis, oxidative phosphorylation, and amino acid metabolism showed no significant (p<0.05) differencesdecreases in the hexose sugars and hypoxanthine by 15% and 17% respectively in cooled skeletal muscle tissue. CONCLUSION: A clinically relevant administration of cryotherapy does not seem to have a significant impact on the transcriptome nor metabolome of otherwise healthy skeletal muscle. Supported by NIH grant U24-DK097153.