Alzheimer's disease (AD) yields a dramatic burden on patients and their families, with no complete cure yet. Our group has previously found that AD-related cognitive impairment (ARCI) could be alleviated after luteolin and exercise combination treatment (Lut + Exe), but the potential mechanisms require further exploration.This work used untargeted metabolomics to uncover the mechanisms Lut + Exe protects against ARCI.Utilizing an Aβ1-42-oligomers-induced AD model, the Morris water maze (MWM) test was performed. Metabolomics of plasma was performed to identify differential metabolites. KEGG and MetaboAnalyst were used to enrich the metabolic pathways. Then, the autophagy inhibitor chloroquine (CQ) was utilized to verify the potential role of autophagy in the Lut+ Exe efficacy against ARCI.The results showed that Lut + Exe alleviated the ARCI in mice. The in-depth analysis showed that Lut + Exe could significantly affect purine metabolism, retinol metabolism, thiamine metabolism, histidine metabolism, and cysteine and methionine metabolism, indicating that the energy metabolism disorder was alleviated. Based on the close relationship between autophagy and energy metabolism, further study found that Lut + Exe could reverse the significant reduction of key autophagy proteins of AD model mice, while the effects of it on the MWM performance and neurogenesis of AD model mice could be blocked by CQ.This study reveals the crucial role of autophagy in the mechanisms of Lut + Exe against ARCI using untargeted metabolomics. Our work provides a novel paradigm to promote the use of combination treatment in curing AD.Copyright © 2025. Published by Elsevier Inc.