Many food items included in the Mediterranean diet (MedDiet) are rich in polyamines, small aliphatic amines with potential cardioprotective effects. The consumption of a MedDiet could increase polyamine concentrations. Based on experimental models, polyamine concentrations may be also influenced by physical activity (PA). We aimed to evaluate whether an intervention based on an energy-restricted MedDiet (er-MedDiet) and PA promotion, in comparison with an energy-unrestricted MedDiet and traditional health care, influences the serum pattern of polyamines and related metabolites in subjects at high risk of cardiovascular disease (CVD). This was a substudy from the PREDIMED-Plus trial, an ongoing randomized clinical trial including 6874 participants allocated either to an intensive weight-loss lifestyle intervention based on er-MedDiet, PA promotion, and behavioral support (er-MedDiet + PA group), or to an energy-unrestricted MedDiet and traditional health care group (MedDiet group). A total of 75 patients (n = 38, er-MedDiet + PA group; n = 37, MedDiet group) were included in this study. Serum concentrations of arginine, ornithine, polyamines, and acetyl polyamines at baseline and 26 wk of intervention were measured by an ultra-high-performance LC–tandem MS platform. At week 26, study groups had similar adherence to the MedDiet but patients randomly assigned to the er-MedDiet + PA group showed significantly lower mean energy intake (−340.3 kcal/d; 95% CI: −567.3, −113.4 kcal/d; P = 0.004), higher mean PA (1290.6; 95% CI: 39.9, 2541.3 metabolic equivalent tasks · min/d; P = 0.043), and higher mean decrease in BMI (in kg/m2) (−1.3; 95% CI: −1.8, −0.6; P < 0.001) than the MedDiet group. However, no significant differences in serum polyamines or related metabolites were found between study groups after 26 wk of intervention and no significant between-group differences were found in glycated hemoglobin, HDL-cholesterol, or triglyceride concentrations. In individuals at high CVD risk, an er-MedDiet with increased PA did not result in significant changes of serum concentrations of polyamines or related metabolites in comparison with an energy-unrestricted MedDiet and no increase in PA. This trial was registered at isrctn.com as ISRCTN89898870. The Mediterranean diet is a healthy dietary pattern that emphasizes the consumption of vegetables, fruits, whole grains, fish, and unsaturated fats such olive oil and nuts, and aims to limit the consumption of saturated fats found in butter and read meats, and sweets. Multiple studies have reported evidence for an inverse relationship between adherence to Mediterranean diet and risk of cardiovascular disease-related mortality associated to the metabolic syndrome. Previous studies on nutritional metabolomics have shown that a higher adherence to the Mediterranean diet is associated to an increase in specific metabolites in the circulation. Remarkably, many food items included in the Mediterranean diet such as fruits, vegetables, legumes, whole grains, fish and nuts are rich in polyamines such as spermidine and spermine, small molecules with potent anti-aging and cardioprotective effects. In this work we have evaluated whether an intervention based on an energy-restricted MedDiet and physical promotion, in comparison with an energy-unrestricted MedDiet and traditional health care, influences the serum pattern of polyamines and related metabolites in subjects at high cardiovascular risk.