ABSTRACTReplacement of muscle tissue by fat in patients with Becker muscular dystrophy (BMD), as measured by quantitative muscle MRI, has been shown to reflect disease progression, but this process is considered irreversible. To monitor treatment effects in healthy‐appearing muscles, biomarkers reflecting disease activity are needed. Here, we compare several candidate biomarkers for disease activity between patients with BMD and controls: intracellular ionised magnesium ([Mg2+]), phosphodiesters (PDE), and weighted pH measures from phosphorus‐(31P)‐MRS; and membrane permeability derived from the random permeable barrier model (RBPM), as applied to diffusion‐tensor‐(DT)‐MRI data. We performed 7 T 31P‐MRS and 3 T DT‐MRI in the left lower leg of 23 participants with BMD (mean [range] age: 41.1 [18.8–66.2] years) and 14 healthy controls (mean [range] age: 43.0 [21.2–63.6] years), estimating [Mg2+], PDE/γ‐ATP, weighted pH and RPBM permeability. Follow‐up scans at 24 months were performed in a subset of these participants. Muscles in patients with BMD were regarded as likely to be ‘preserved’ (BMDpre) with fat fractions ≤ 13.5% and as ‘progressing’ (BMDprog) with 13.5% < fat fraction < 81.5%. Muscles with fat fractions ≥ 81.5% were excluded from further analyses. We observed decreased [Mg2+] in BMDpre and BMDprog compared to healthy controls, whereas PDE/γ‐ATP and weighted pH were increased in these muscles. RBPM‐measured permeability did not differ between groups. We observed no longitudinal changes in [Mg2+], PDE/γ‐ATP or weighted pH. Only [Mg2+] continued to show group differences on inclusion of longitudinal data, and weighted pH demonstrated inter‐muscle differences. In patients with BMD, 31P‐MRS demonstrates reduced [Mg2+] and increased weighted pH in the lower leg muscles versus controls, suggesting greater membrane permeability—a potential disease activity biomarker independent of the disease phase. PDE/γ‐ATP was also significantly increased in progressing and preserved muscle. Incorporating 31P‐MRS in therapeutic trials will help to further establish its use as a response biomarker.