Despite the recognized relevance of physical activity in the development and health of children, assessment of physical activity levels (PA-level) in infancy is limited, especially in infants with Down syndrome (DS). Increasing PA-level using a treadmill training (TMT) intervention may benefit infants with motor delays. The purpose of this study was to investigate whether a higher intensity, individualized TMT protocol (HI) would elicit immediate and short-term higher PA-level in infants with DS than a lower intensity, generalized training protocol (LG). Thirty infants with DS were randomly assigned to the LG or the HI group. Training was terminated when the infants could walk three steps independently. Activity monitors were placed on infants' trunks and legs for a 24-h period every other month during the intervention phase, and at set intervals 1-year post-independent walking onset. Data were analyzed to separate sedentary-to-light activity (Lowact) and moderate-to-vigorous activity (Highact). Overall our results demonstrate that infants receiving the HI TMT had higher levels of Highact than infants in the LG group, factoring out the activity produced by the intervention itself. Infants in the LG group spent more time in Lowact than the HI group. In addition, these results seemed to be retained during the post-intervention follow-up. Despite these exciting results, more work is needed to understand the relationship of the intensity of TMT on PA-level in infants with DS, and whether PA-level and TMT are related to motor milestone achievement and/or health benefits.