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BACKGROUND In order to cope with adverse environmental conditions, plants activate defense mechanisms leading to the accumulation and/or depletion of general and specialized metabolites. In this study, a multiplatform untargeted metabolomics strategy was employed to evaluate metabolic changes in strawberry fruit of cv. Camarosa grown under osmotic stress conditions. Liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry data from strawberry grown under two water deficit conditions, irrigated at 95% crop evapotranspiration (ETc) and 85% ETc, and one excess salt condition with a 80 mM NaCl solution were analyzed to determine treatment effects on fruit metabolism. RESULTS Multivariate principal component analysis, orthogonal projections to latent structures - discriminant analysis, and univariate statistical analyses were applied to the data set. While multivariate analyses showed group separation by treatment, T-test and fold-change revealed twelve metabolites differentially accumulated in strawberries from different treatments, among them phenolic compounds, glycerophospholipids, phytosterols, carbohydrates and an aromatic amino acid. CONCLUSION Untargeted metabolomic analysis allowed for the annotation of compounds differentially accumulated in strawberry fruit from plants grown under osmotic stresses and non-stressed plants. The metabolic disturbance in plants under stress involved metabolites associated with inhibition of reactive oxygen species and cell wall and membrane lipid biosynthesis, which might serve as osmotic stress biomarkers. This article is protected by copyright. All rights reserved.