Background: The aim of this study was to examine the bidirectional longitudinal associations of several markers of fatness and physical fitness in adolescents with Down syndrome. Methods: This study comprised a total of 111 adolescents (41 females), aged from 11 to 20 years with complete data at the baseline. We had a drop-out of <10% from the baseline to the 2-year follow-up. The ALPHA health-related fitness test battery for youth was used. Results: Our results show that all fatness variables at the baseline were associated with a 2-year change in cardiorespiratory fitness ([beta] ranging from -0.32 to -0.38; all p < 0.05), but not with muscular and motor fitness (p > 0.05). However, no associations were found between physical fitness components as predictors and fatness indicators (p > 0.05). Conclusions: Results suggest that reducing fatness during adolescence might represent a modifiable factor to improve cardiorespiratory fitness at the 2-year follow-up, but not vice versa since associations were not bidirectional. [This article was written on behalf of the UP, DOWN study group.]