Abstract Objectives We aimed to characterize the effects of a low dose of a sustained-release L-arginine (L-ARG) supplementation on plasma metabolome in healthy overweight adults with cardiometabolic risk factors, and study if its effect could improve the capacity to handle a high fat-high sugar meal, that deeply and acutely challenge metabolic capacities. Methods In a randomized, double-blind, controlled trial, 33 healthy overweight adults (BMI > 25kg/m²) with cardiometabolic risk (plasma triglycerides > 150 mg/dL; waist circumference > 94 cm (men) or > 80 cm (women)) were treated with 1.5 g L-ARG 3 times/d (4.5 g/d) or placebo for 4 weeks. On the last day of treatment, the volunteers consumed a high fat, high sugar meal challenge (900 kcal). Plasma was collected at fasting and 2, 4 and 6 h after the meal. Metabolites were analyzed by high-resolution mass spectrometry hyphenated to liquid chromatography, using reversed and normal phase columns, and operated in both positive and negative ionization modes. Annotation was performed using an in-house database referencing more than 1300 metabolites. Metabolic profiles were analyzed using Linear Mixed Models-PCA (LiMM-PCA), which enables to analyze repeated multivariate data. LiMM-PCA combines linear mixed models and PCA to assess the effects of both fixed factors (Treatment, Time before and after meal, interaction Treatment x Time, Order of treatment administration and Period of the study) and random factors (Individual). Results 521 metabolic features were identified in the plasma. Time accounted for most part of the variance in dataset (30.0%, P < 0.001), showing that the challenge meal leaved the largest imprint on the metabolome, with a clear distinction between fasting and fed states. In contrast, interindividual variability explained 10.1% of the total variance. L-ARG treatment significantly affected plasma metabolome (P < 0.001), explaining 0.6% of the variance. This effect did not differ after the challenge meal (time–treatment interaction, P > 0.10). The analysis of the metabolites contributing the most to these effects is under progress. Conclusions L-ARG supplementation has a significant effect on the metabolome in a situation where the metabolome is heavily impacted by a large allostatic load, and these two effects are independent of each other. Funding Sources Supported by a grant from Pierre Fabre Research Institute.