SCOPE: Phenolic compounds are minor components of extra virgin olive oil (EVOO). Secoiridoids are the major components contributing to the phenolic content of EVOO. {Silva, 2015 #95}Information is lacking regarding their potential as biomarkers for EVOO intake. METHODS AND RESULTS: Healthy volunteers (n = 9) ingested 50 mL of EVOO in a single dose containing 322 mg/kg total phenolic content (caffeic acid equivalents) and 6 mg/20g hydroxytyrosol and its derivatives. Plasma was collected before (0h) and at 0.5, 1, 2, 4 and 6h after ingestion. Urine samples were collected prior to ingestion (0h) and at 0-4, 4-8, 8-15 and 15-24h. Samples were analysed by UPLC coupled with an Exactive Orbitrap MS. Partial Least Squares Discriminant Analysis with Orthogonal Signal Correction was applied to screen for metabolites that allowed samples discrimination. Plasma biomarkers and urine biomarkers were selected although individual variability was observed among volunteers. Results are in accordance with in vitro experiments performed (in vitro digestion and hepatic microsomal activity assays). CONCLUSIONS: Plasma (elenolic acid + H2 ; p-HPEA-EA + H2 + glucuronide) and urinary (3,4-DHPEA-EA, 3,4-DHPEA-EA + H2 +glucuronide, methyl 3,4-DHPEA-EA + H2 +glucuronide) secoiridoid compounds were selected as biomarkers to monitor EVOO intake showing good predictive ability according to multivariate analysis.