Using sub-microlitre voxel size in magnetic resonance spectroscopy (MRS) is advantageous to investigate specific small brain region without any partial volume effect. The aim of this study was to determine the intra-individual coefficient-of-variation of neurochemical concentrations when using sub-microlitre MRS voxel measured during 3 consecutive days. This study was performed in adult mice at 11.7T. The intra-individual CV for the major and high concentration brain metabolites was ≤ 9% for MRS data collected in pre-clinically feasible scan times. This in turn shows that sub-microlitre can be used in pre-clinical application to detect subtle changes in neurochemical profile in diseased brain.