Staphylococcus aureus is the causative agent of a high number of acute and chronic diseases due to its extraordinary capacity to rapidly adapt to changes in environmental conditions. This study has used prolonged cold treatment as a model system to explore changes in the proteome and metabolomic following environmental challenges. Metabolic and proteomic profiles were generated from cytoplasmic extracts from cells harvested at the mid exponential phase of growth under ideal conditions at 37ºC. These were used for comparison with equivalent sets of cells from the same stock which were subsequently exposed to prolonged cold stress for 2 weeks at 4 °C. Principle component analysis (PCA) showed that prolonged cold stress conditions generated cells with substantially different profiles of cytoplasmic amino acids and proteins compared with reference controls (P<0.05). All replicates from the prolonged cold treatment were closely clustered and well separated from the control replicates. The total concentration of cytoplasmic amino acids was reduced by 75% in the cells exposed to cold compared with the controls. It was noted that citric acid was the major metabolite measured in the cytoplasm from the cells exposed to cold, where it was virtually absent in the control extracts. The results indicated an unexpected increase in production of nine ribosomal proteins (S1, S7, S8, L3, L5, L10, L24, L25, and L30) in the cytoplasmic fractions in the cells exposed to cold-stress. The data provided strong evidence that specific alterations in amino acid and protein compositions were associated with the adaptive response to facilitate survival under the exposure to prolonged cold conditions.