Objective This study aimed to measure the impact of a school-based multidisciplinary intervention program on risk factors for atherosclerosis in sixth-grade middle school students. We also measured health behaviors before and after the intervention using a validated questionnaire. Methods A prospective study was performed in which students served as historical controls. Sixth-grade students from 23 middle schools in 12 cities with varying populations were exposed to a program promoting healthful activity and nutrition habits through educational and environmental change. Along with a modified School-Based Nutrition Monitoring behavioral questionnaire, physiologic risk factors were studied, including body mass index, systolic and diastolic blood pressures, cholesterol panel, and random blood glucose, which were measured before the 10-week program and again 1 to 3 months after program completion. Results Of 4021 sixth graders (male, 49%) at 23 middle schools completing a before and after behavioral survey, 2118 students, aged 11.56 ± 0.47 years, consented to participate in the screening. The mean total cholesterol value decreased from 161.64 ± 28.99 mg/dL to 154.77 ± 27.26 mg/dL (P <.001). The low-density lipoprotein value decreased from 89.37 ± 25.08 mg/dL to 87.14 ± 24.25 mg/dL (P <.001). The high-density lipoprotein value decreased from 52.15 ± 13.35 md/dL to 49.95 ± 13.28 mg/dL (P <.001). The measure of triglycerides decreased from 113.34 ± 73.19 mg/dL to 101.22 ± 63.93 mg/dL (P <.001). The random glucose value decreased from 97.51 ± 16.00 to 94.94 ± 16.62 (P <.001). The mean systolic blood pressure decreased from 109.47 ± 15.26 mm Hg to 107.76 ± 10.87 mm Hg (P <.001), and the mean diastolic blood pressure decreased from 64.78 ± 8.57 mm Hg to 63.35 ± 7.81 mm Hg (P <.001). These changes in physiologic measures seemed to correlate with self-reported increases in vegetable and fruit consumption, increases in physical activity, and less screen time. Conclusions Project Healthy Schools, a middle school intervention to improve childhood cardiovascular risk factors, is feasible and seems to be effective. The results showed significant improvements in risk factors associated with early atherosclerosis among sixth-grade students, including total cholesterol, low-density lipoprotein cholesterol, triglycerides, and systolic and diastolic blood pressures. Further study with a larger group of students and a longer follow-up period would be valuable.