Design of clinical trials and evaluations of treatment efficacy in DMD require an understanding of the meaningfulness of changes in functional measures. An important concept is the minimal detectable change (MDC), i.e., the minimal magnitude of measured change indicative of true change due to disease progression or maturation, rather than transient variation or measurement error. We used data from six natural history studies and eight clinical trial placebo arms to obtain MDC estimates for North Star Ambulatory Assessment (NSAA) total (n=1,325), 4-stair climb (4SC) completion time and velocity (n=1,274), and 6-minute walk distance (6MWD; n=777). Patients aged ≥4 to <18 years, receiving steroids, and with at least minimal baseline ambulatory function (NSAA > 12, 4SC time < 12 seconds, or 6MWD > 75 meters) were analyzed. Variation around patient-specific fitted trajectories, based on longitudinal mixed effects models, was used to estimate MDCs and thresholds that provide >80% confidence that observed changes reflect underlying definitive functional change. Minimal important difference estimates based on 0.5 standard deviation (SD) of baseline values were also calculated. Estimated thresholds for >80% confidence in true change were 2.8 units for NSAA, 2.2 seconds for 4SC time, 0.36 stairs/second for 4SC velocity and 36.3 meters for 6MWD. MDC estimates were smaller in natural history than in placebo arm data for 4SC time (1.9 vs. 2.6 seconds, reflecting sensitivity to differences in maximum completion times recorded, which were typically higher in trials), but similar across natural history and trials for other measures. Thresholds for minimal detectable change in NSAA, 4SC time and 6MWD were larger among patients with lower function, but similar across different age groups. MDC estimates were smaller than 0.5 SD estimates. The identified thresholds can be used to inform endpoint definitions, as inputs into power calculations, or as benchmarks to contextualize individual and group-level changes due to treatment.