The Mediterranean diet (MD) is considered a dietary pattern with beneficial effects on human health. The aim of this study was to assess the effect of an MD on urinary metabolome by comparing subjects at 1 and 3 years of follow-up, after an MD supplemented with either extra-virgin olive oil (MD+EVOO) or nuts (MD+Nuts), to those on advice to follow a control low-fat diet (LFD). Ninety-eight non-diabetic volunteers were evaluated, using metabolomic approaches, corresponding to MD+EVOO (n=41), MD+Nuts (n=27) or LFD (n=30) groups. The 1 H-NMR urinary profiles were examined at baseline, after 1 and 3 years of follow-up. Multivariate data analysis (OSC-PLS-DA and HCA) methods were used to identify the potential biomarker discriminating groups, exhibiting a urinary metabolome separation between MD groups against baseline and LFD. Results revealed that the most prominent hallmarks concerning MD groups were related to the metabolism of carbohydrates (3-hydroxybutyrate, citrate, cis-aconitate), creatine, creatinine, amino acids (proline, Nacetylglutamine, glycine, branched-chain amino acids and derived metabolites), lipids(oleic and suberic acids), and microbial co-metabolites (phenylacetylglutamine, pcresol). Otherwise, hippurate, trimethylamine-N-oxide, histidine and derivates (methylhistidines, carnosine, anserine) and xanthosine were predominant after LFD. The application of NMR-based metabolomics enabled the classification of individuals regarding their dietary pattern and highlights the potential of this approach for evaluating changes in the urinary metabolome at different time points of follow-up in response to specific dietary interventions.2