Background <p>Higher LDL cholesterol levels are associated with increased risk of cardiovascular disease, and early intervention in children and regular follow-up are keystones for its prevention. Hypercholesterolemia can be partially delayed adopting healthy lifestyles, including healthy diets such as the Mediterranean diet and physical activity. Extra-virgin olive oil (EVOO), the main edible oil in the Mediterranean diet, has demonstrated cardiovascular benefits.</p> Objective <p>The present randomized double-blind crossover clinical study aimed to evaluate the efficacy of the daily consumption of a spreadable cream based on extra virgin olive oil enriched in phytosterols (0.04–0.06&#xa0;g plant sterols/kg of body weight/day) for 8&#xa0;weeks separated by a wash-out period of 4&#xa0;weeks on children (6–18&#xa0;years old) with hypercholesterolemia.</p> Methods <p>Fifty children were included although only 23 reported at least 70% of compliance and were used for final analyses. We used a dual approach combining the determination of anthropometric and biochemical plasma parameters and the lipidomics profile of plasma samples.</p> Results <p>Spreadable cream enriched in phytosterols consumption led to a decrease in total cholesterol and low-density lipoprotein cholesterol plasma levels. Multivariate analysis of the lipidomic plasma profile indicated that phospholipids and sphingolipids were mostly involved in modulating the observed effects.</p> Conclusions <p>Overall, this study provides a multi-level integrated approach for the characterization of a novel functional food to counteract hypercholesterolemia from a nutritional perspective.</p> Clinical Trial Registry <p>Clinical Trial Registry number and website where it was obtained <a href="https://clinicaltrials.gov/ct2/show/NCT05460208">https://clinicaltrials.gov/ct2/show/NCT05460208</a></p>