The potential benefits of tomato-rich diets for the cardiovascular system have been related to plasma concentrations of carotenoids. In addition, the bioavailability of carotenoids from foods depends on their chemical structure, processing and the food matrix. Our aim was to evaluate the effect of adding oil to tomato juice (not treated with heat) on the bioavailability of plasma carotenoids and postprandial lipid response. In a randomized, controlled, crossover feeding trial, eleven healthy volunteers were assigned to receive a single ingestion of 750 g of tomato juice (TJ) containing 10% of refined olive oil/70 kg body weight (BW) and 750 g of TJ without oil/70 kg BW on two different days. All lycopene isomers increased significantly in subjects consuming TJ with oil, reaching the maximum concentration at 24 h. LDL cholesterol and total cholesterol decreased significantly 6 h after the consumption of TJ with oil, which significantly correlated with an increase of trans-lycopene and 5-cis-lycopene, respectively.