Diet-induced effects on the intestinal and systemic metabolome upon adherence to the Mediterranean diet (Med-D) may be beneficial for maintaining cardiovascular health. To investigate the effects of the Med-D compared to habitual diet on the intestinal and systemic metabolome, we conducted a randomized controlled parallel trial in 82 overweight or obese men and women with no known history of cardiovascular disease. Each subject in the Med-D group was assigned a personalized diet prepared on the basis of his/her habitual diet. Energy values and whole macronutrient composition were unchanged during the Med-D intervention. Feces, urine and blood samples were collected from all participants at baseline, after 4 and 8 weeks intervention. The samples were subjected to untargeted metabolomics using ultra-performance liquid chromatography time-of-flight mass spectrometry (UPLC-MS). Among the metabolites changing with the Med-D, we identified several food biomarkers confirming compliance including reduced levels of carnitine (reflecting reduced meat intake), increased levels of alkylresorcinols (reflecting increased wholegrain intake), increased levels of Urolithin A-3-O-glucuronide (reflecting increased nut intake). Furthermore, the Med-D resulted in reduced levels of the host-microbial co-metabolite p-cresol-sulfate (reflecting reduced colonic proteolysis) as well as changes in the primary bile acid, chenodeoxycholic acid-3-sulfate (suggesting altered bile acid biosynthesis and/or excretion), and reduced acyl-carnitines (possibly reflecting reduced beta-oxidation). Finally, the Med-D resulted in a changed tryptophan metabolism as reflected by alterations in both microbially derived tryptophan catabolites as well as in host tryptophan metabolites. Collectively, these alterations confirm that a Med-D changes the intestinal and systemic metabolome by changing circulating levels of food biomarkers, microbial metabolites and host metabolites.