Background: Protective factors associated with positive youth development predict health and education outcomes. This study explored trends in these protective factors and in physical activity among low-income students, and determined the impact of a school-based youth development program on these trends. Methods: This study used a quasi-experimental time series design including data from 158 low-income schools from 2001 to 2007. Ninety-four schools had exposure to a school-based program promoting physical activity and youth development through structured play; 64 schools served as controls. Primary outcomes were 5th-grade student scores (n = 13,109) on a California statewide survey for physical activity (1-6 scale) and measures of protective factors including problem solving skills, meaningful participation in school, and caring adults (1-4 scales). Predictors were time (year) and school's number of years of exposure to the program. Results: Overall, significant annual declines were seen in protective factors, including students' report of feeling safe (-0.03, 95% CI [-0.03, -0.01]), caring adults at school (-0.03 [-0.05, -0.02]), and problem solving skills (-0.03 [-0.04, -0.02]). Cumulative declines over 6 years were equivalent to a drop of 1 school-level SD. Each additional year of exposure to the program predicted greater meaningful participation (0.02 [0.001, 0.5]), problem-solving skills (0.03 [0.0001, 0.06]), and increased physical activity (0.06 [0.01, 0.10]); exposure throughout elementary school (6 years) increased scores by 1 school-level SD. Conclusions: Low-income students reported a significant decline in protective factors since 2001. School partnerships with youth development programs promoting physical activity may ameliorate declines in emotional well-being and increase physical activity. (Contains 2 figures and 3 tables.)