The Mediterranean diet, a traditional dietary pattern of the Mediterranean region, encompasses numerous cultural variations. The dietary intake from the rural area of Apulia, Italy has been collected in a population-based cohort study for the past two decades. A study was conducted in this cohort to describe and analyse dietary intake in association with biomarker profiles. The study population showed remarkable stability across dietary intake, particularly high intakes of vegetables, fruit, and local pasta, as well as anthropometric measures, blood pressure, and biochemical markers. Furthermore, with the use of repeated measures, olive oil was inversely correlated with HDL-cholesterol.