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Objective: To perform a comprehensive lipid profiling to evaluate potential lipid metabolic differences between patients with amyotrophic lateral sclerosis (ALS) and controls, and to provide a more profound understanding of the metabolic abnormalities in ALS. Methods: Twenty patients with ALS and 20 healthy controls were enrolled in a cross-sectional study. Untargeted lipidomics profiling in fasting serum samples were performed by optimized UPLC–MS platforms for broad lipidome coverage. Datasets were analyzed by univariate and a variety of multivariate procedures. Results: We provide the most comprehensive blood lipid profiling of ALS to date, with a total of 416 lipids measured. Univariate analysis showed that 28 individual lipid features and two lipid classes, triacylglycerides and oxidized fatty acids (FAs), were altered in patients with ALS, although none of these changes remained significant after multiple comparison adjustment. Most of these changes remained constant after removing from the analysis individuals treated with lipid-lowering drugs. The non-supervised principal component analysis did not identify any lipid clustering of patients with ALS and controls. Despite this, we performed a variety of linear and non-linear supervised multivariate models to select the most reliable features that discriminate the lipid profile of patients with ALS from controls. These were the monounsaturated FAs C24:1n-9 and C14:1, the triglyceride TG(51:4) and the sphingomyelin SM(36:2). Conclusions: Peripheral alterations of lipid metabolism are poorly defined in ALS, triacylglycerides and certain types of FAs could contribute to the different lipid profile of patients with ALS. These findings should be validated in an independent cohort. [ABSTRACT FROM AUTHOR]