Dietary strategies to improve early cardiovascular markers in overweight children are needed. We investigated the effect of dietary protein and glycemic index (GI) on cardiovascular markers and metabolic syndrome (MetS) scores in 5- to 18-y-old children of overweight/obese parents from 8 European centers. Families were randomized to 1 of 5 diets consumed ad libitum: high protein (HP) or low protein (LP) combined with high GI (HGI) or low GI (LGI), or a control diet. At 6 centers, families received dietary instruction (instruction centers); at 2 centers, free foods were also provided (supermarket centers). Diet, anthropometry, blood pressure, and serum cardiovascular markers (lipid profile, glucose regulation, and inflammation) were measured in 253 children at baseline, 1 mo, and/or 6 mo. Protein intake was higher in the HP groups (19.9 ± 1.3% energy) than in the LP groups at 6 mo (16.8 ± 1.2% energy) (P = 0.001). The GI was 4.0 points lower (95% CI: 2.1, 6.1) in the LGI compared with the HGI groups (P <0.001). In the supermarket centers, the HP and LP groups differed more in protein intake than did the groups in the instruction centers (P = 0.009), indicating better compliance. The HP diets evoked a 2.7-cm (95% CI: 0.9, 5.1) smaller waist circumference and a 0.25-mmol/L (95% CI: 0.09, 0.41) lower serum LDL cholesterol compared with the LP diets at 6 mo (P <0.007). In a separate supermarket center analysis, the HP compared with LP diets reduced waist circumference (P = 0.004), blood pressure (P <0.01), serum insulin (P = 0.013), and homeostasis model of assessment-insulin resistance (P = 0.016). In the instruction centers, the HP compared with the LP diets reduced LDL cholesterol (P = 0.004). No consistent effect of GI was seen and the MetS scores were not affected. In conclusion, increased protein intake improved cardiovascular markers in high-risk children, particularly in those undergoing most intensive intervention.