Background: North Star Ambulatory Assessment (NSAA) is used to measure functional performance in Duchenne Muscular Dystrophy (DMD). An evaluation of decline in skeletal muscle quality and its effect on NSAA performance is warranted to better understand the potential clinical trial outcomes at different ages in DMD. Dixon based MRI measures muscle fat fraction (FF) and is a sensitive outcome to monitor disease progression in DMD. This study performed a comprehensive analysis of lower extremity muscles FF and performance in NSAA at various ages. Method: Three-year longitudinal data (NSAA and FF) were acquired from boys with DMD (age 5-18 years, n=50). Fat fraction maps were generated from Dixon images acquired using a 3T MRI system at multiple sites. Longitudinal changes in NSAA score were compared between groups defined by age and FF measured in individual muscles as well as composite FF. Statistical analysis was performed using repeated measures ANOVA, Spearman correlation, and standardized response mean (SRM). Results: As age increases, FF increases significantly in all lower extremity muscles, except the tibialis anterior and posterior. A nonlinear decline in NSAA with age was observed, with an average annual decline of 3 points. A rapid decline in NSAA (average decline: 4-12 points over 3 years) was observed between 8-15 years of age. NSAA significantly correlated with lower extremity muscle FF and the greatest correlation was observed with vastus lateralis FF (?= -0.82, p<0.001). Over 3-years, participants with baseline composite FF of ?0.20 to ?0.40 showed the greatest decline in NSAA (average decline= 5-8 points; SRM= 1.27-2.03). When composite FF reached 0.40, >75 percentile of cohort had lost their ability to complete ambulatory skills of NSAA. Conclusion: Findings demonstrate an inverse relationship between lower extremity muscle FF and NSAA, and decline in NSAA depends on patient’s baseline FF. These findings hold important implications for designing clinical trials.