We previously reported that calcium intake enhanced the leg bone response to physical activity of preschool children in a 12-month randomized trial of calcium supplementation and physical activity. To determine whether the intervention-induced changes in leg bone mineral content and size were maintained through the subsequent 12-month follow-up period, total body bone measurements by DXA and 20% distal tibia pQCT bone measurements were obtained at 24 months (12 months post-intervention). Children also were measured for height and weight, and accelerometer readings were obtained in a subset of children at 18 and 24 months (6 and 12 months post-intervention). Regression analyses were performed controlling for covariates and indicated that increases from 12 to 24 months were greater in the gross motor (GM) activity group (bone loading, large muscle exercises) vs. fine motor (FM) activity group (arts and crafts program) for arm bone area (BA) (P <0.01), total body (P=0.04) and arm (P <0.01) bone mineral content (BMC). There were no differences in BA or BMC changes from 12 to 24 months by calcium supplementation. Differences in tibia periosteal circumference by pQCT persisted at 24 months (GM 51.4 +/- 0.4 mm vs. FM 50.2 +/- 0.4 mm, P=0.03) with a trend for greater endosteal circumferences in the children in the GM vs. FM groups at both 12 and 24 months (both, P=0.08). There were no significant differences in cortical area or thickness by activity or supplement group at 24 months. Children in the GM group had greater accelerometer counts/day (P=0.04) and more time in vigorous activity (P=0.05) at 18 months compared to FM group. No differences in accelerometer readings were noted at 24 months. In conclusion, we found higher activity levels in children randomized to gross motor vs. fine motor activities 6 months after the intervention program ceased. Whether the greater periosteal circumference that was observed 12 months post-intervention was a persistent biological bone effect or due to persistently higher activity levels is not known.