It was aimed to detect the novel future biomarkers using a metabolomics approach in premature calves. Calves born previous to 270 days' pregnancy are at risk, and the earlier the calving is, the higher the risk. More trials are needed in neonatology field as it little known almost the generally metabolic status of preterm neonates. To date, this is the first NMR (nuclear magnetic resonance) based study on serum metabolomics at set intervals in premature calves. Biochemical health profiles and NMR based metabolomic analysis were performed in twenty-five premature dairy calves. The whole animals partly recovered following 72h. Clinical data were compatible with those of premature animals. Increased levels of AST and CPK may be attributed to subclinic trauma at birth. Alterations in metabolites, increases in 3-hydroxybutyrate, citrate, leucine and isoleucine at 48th and 72h; choline, formate, fatty acids and polyunsaturated fatty acids at 72h, and valine at 48h; and decreases in myo-inositol at 48h and 72h were meaningful for monitoring the recovery at a molecular level in premature calves. Metabolomics became an important tool for identification of premature calves’ clinical pathology and monitoring therapeutic picture.