Background: Nerve growth factor (NGF) and brain-derived neural factor (BDNF) are important for brain function and detectable in the blood. This study explored the longitudinal associations of physical activity and body fat with serum NGF and BDNF in children. Methods: Two waves of measurements were performed 2 years apart in 8- to 11-year-old children, including physical activity using the ActiGraph model 7164, body composition by dual-energy X-ray absorptiometry, and serum NGF and BDNF determined by multiplex immunoassay. The first wave included 248 children. Full information maximum likelihood estimation with robust standard errors was applied in structural equation modeling. Results: Vigorous physical activity showed a direct positive longitudinal relationship with NGF (standardized coefficient β = 0.30, P = .01) but not with BDNF (β = 0.04, P = .84). At the same time, body fat percentage was positively related to both NGF (β = 0.59, P < .001) and BDNF (β = 0.17, P = .04). There was an indication of an indirect relationship of vigorous physical activity with NGF (product of unstandardized coefficient β = −0.18, P = .02) and BDNF (β = −0.07, P = .05) through the negative relationship with body fat percentage (β = −0.36, P < .001). Conclusions: Vigorous physical activity is directly related to serum NGF and indirectly through the level of body fat. The relationships with serum BDNF are more complex.