Objective: Sensitive, objective, and easily applied methods for evaluating disease progression and response to therapy are needed for clinical trials in Duchenne muscular dystrophy (DMD). In this study, we evaluated whether electrical impedance myography (EIM) could serve this purpose. Methods: In this nonblinded study, 36 boys with DMD and 29 age-similar healthy boys underwent multifrequency EIM measurements for up to 2 years on 6 muscles unilaterally along with functional assessments. A linear mixed-effects model with random intercept and slope terms was used for the analysis of multifrequency EIM values and functional measures. Seven DMD boys were initiated on corticosteroids; these data were analyzed using a piecewise linear mixed-effects model. Results: In boys > 7.0 years old, a significant difference in the slope of EIM phase ratio trajectories in the upper extremity was observed by 6 months of −0.074/month, p = 0.023, 95% confidence interval (CI) = −0.013, −0.14; at 2 years, this difference was −0.048/month, p < 0.0001, 95% CI = −0.028, −0.068. In boys ≤ 7.0 years old, differences appeared at 6 months in gastrocnemius (EIM phase slope = −0.83 °/kHz/mo, p = 0.007, 95% CI = −0.26, −1.40). EIM outcomes showed significant differences earlier than functional tests. Initiation of corticosteroids significantly improved the slope of EIM phase ratio (0.057/mo, p = 0.00019, 95% CI = 0.028, 0.086) and EIM phase slope (0.14 °/kHz/mo, p = 0.013, 95% CI = 0.028, 0.25), consistent with corticosteroids' known clinical benefit. Interpretation: EIM detects deterioration in muscles of both younger and older boys by 6 months; it also identifies the therapeutic effect of corticosteroid initiation. Because EIM is rapid to apply, painless, and requires minimal operator training, the technique deserves to be further evaluated as a biomarker in DMD clinical therapeutic trials. Ann Neurol 2017;81:622–632.