Brain metabolism evolves rapidly during early post-natal development in the rat. While changes in amino acids, energy metabolites, antioxidants or metabolites involved in phospholipid metabolism have been reported in the early stages, neurometabolic changes during the later post-natal period are less well characterized. Therefore, we aimed to assess the neurometabolic changes in male Wistar rats between post-natal days 29 and 77 (p29-p77) using longitudinal magnetic resonance spectroscopy (MRS) in vivo at 9.4 Tesla. ¹ H MRS was performed in the hippocampus between p29 and p77 at 1-week intervals (n = 7) and in the cerebellum between p35 and p77 at 2-week intervals (n = 7) using the SPECIAL sequence at ultra-short echo-time. NOE enhanced and ¹ H decoupled ³¹ P MR spectra were acquired at p35, p48 and p63 (n = 7) in a larger voxel covering cortex, hippocampus and part of the striatum. The hippocampus showed a decrease in taurine concentration and an increase in glutamate (with more pronounced changes until p49), seemingly a continuation of their well-described changes in the early post-natal period. A constant increase in myo-inositol and choline-containing compounds in the hippocampus (in particular glycero-phosphocholine as shown by ³¹ P MRS) was measured throughout the observation period, probably related to membrane metabolism and myelination. The cerebellum showed only a significant increase in myo-inositol between p35 and p77. In conclusion, this study showed important changes in brain metabolites in both the hippocampus and cerebellum in the later post-natal period (p29/p35-p77) of male rats, something previously unreported. Based on these novel data, changes in some neurometabolites beyond p28-35, conventionally accepted as the cut off for adulthood, should be taken into account in both experimental design and data interpretation in this animal model.