Background: Biochemical changes, which may precede morphological changes, could be of great relevance in profiling preclinical AD. We aimed to investigate metabolic changes using magnetic resonance spectroscopy (MRS) in three AD‐relevant regions: right hippocampus, right angular gyrus, and right precuneus, focusing on myoinositol due to its role in glial activity (osmoregulation‐neuroinflammation).Method: Three hundred sixty‐six participants from the ALFA+ cohort underwent three single‐voxel MRS (PRESS sequence, TR = 2000ms; TE = 100ms chosen to invert myoinositol peak for more precise quantitation; volume of interest (VOI): 20×20×20mm3 [25×20×16mm3 for hippocampus]). Figure 1 shows VOI location and MRS samples. Concentrations of N‐acetyl‐containing compounds (NAA) that is a neuronal marker, total creatine (Cr) related to brain energy, myoinositol (mI), choline‐containing compounds (Cho) related to cell proliferation, and glutamate/glutamine were calculated with LCModel in arbitrary units (using water reference) with the default settings and basis set adapted to the acquisition conditions. Concentrations were corrected for VOI CSF content. Remaining VOI gray matter percentage was used as a confounder in all analyses, jointly with age, sex, and APOE‐e4 status (carrier/non‐carrier). We investigated the correlation of MRS metabolite concentrations with CSF concentrations of Aβ42/40 and ptau‐181 as biomarkers for AD (Elecsys®; Roche Diagnostics). For completeness, we also checked the dependency of MRS metabolites on age. MRS not meeting QA criteria were discarded (Cramer‐Rao lower bounds >20 for any of the metabolites or imprecise VOI location). The statistical threshold was set to p181 levels in the angular gyrus. NAA was negatively associated with age on cortical regions, as well as Cr on the angular gyrus. Table 2 summarizes the main results.Conclusion: MRS allowed determination of differential metabolite changes in AD‐related regions in preclinical AD. Further research is warranted to better characterize the nature of these metabolic changes and determine their impact on longitudinal outcomes of these participants.