BackgroundDiet is one of the most important modifiable lifestyle factors in human health and in chronic disease prevention. Thus, accurate dietary assessment is essential for reliably evaluating adherence to healthy habits.ObjectivesThe aim of this study was to identify urinary metabolites that could serve as robust biomarkers of diet quality, as assessed through the Alternative Healthy Eating Index (AHEI-2010).DesignWe set up two-center samples of 160 healthy volunteers, aged between 25 and 50, living as a couple or family, with repeated urine sampling and dietary assessment at baseline, and 6 and 12 months over a year. Urine samples were subjected to large-scale metabolomics analysis for comprehensive quantitative characterization of the food-related metabolome. Then, lasso regularized regression analysis and limma univariate analysis were applied to identify those metabolites associated with the AHEI-2010, and to investigate the reproducibility of these associations over time.ResultsSeveral polyphenol microbial metabolites were found to be positively associated with the AHEI-2010 score; urinary enterolactone glucuronide showed a reproducible association at the three study time points [false discovery rate (FDR): 0.016, 0.014, 0.016]. Furthermore, other associations were found between the AHEI-2010 and various metabolites related to the intake of coffee, red meat and fish, whereas other polyphenol phase II metabolites were associated with higher AHEI-2010 scores at one of the three time points investigated (FDR < 0.05 or β ≠ 0).ConclusionWe have demonstrated that urinary metabolites, and particularly microbiota-derived metabolites, could serve as reliable indicators of adherence to healthy dietary habits.Clinical Trail Registrationwww.ClinicalTrials.gov, Identifier: NCT03169088.