The aim of this study was to investigate the effect of a 2-year school-based physical activity (PA) intervention in 9-year-old children on cardiovascular disease (CVD) risk factors. One intervention school (I-school) (n=125) and one control school (C-school) (n=131) were included. The children at the I-school carried out 60 min of PA daily. The PA lessons were planned, organized and led by expert physical education (PE) teachers. In the C-school, children were offered the normal 45 min of PE twice weekly. The intervention resulted in a greater beneficial development in systolic (P=0.003) and diastolic (P=0.002) blood pressure, total cholesterol-to-high-density lipoprotein cholesterol ratio (P=0.011), triglyceride (P=0.030) and peak oxygen uptake (P<0.001) in I-school children than in C-school children. No significant differences were observed in waist circumference, body mass index and the homeostasis model assessment for insulin resistance between the two groups. Furthermore, the intervention, primarily carried out at moderate intensity, had the strongest impact in children with the least favorable starting point. In conclusion, a daily school-based PA intervention can beneficially modify children's CVD risk profile if the intervention has sufficient duration and includes a substantial amount of daily PA, and if the PA is implemented by expert PE teachers.