Immobilization results in muscle atrophy and reversing this presents a challenge to rehabilitation medicine. Functional loss is greater than that suggested by loss of cross-sectional area but assessment is difficult while in a cast. Our aim was to investigate the use of magnetic resonance imaging (MRI) to generate imaging biomarkers of muscle function. We report here the results of cast immobilization on MRI measurements of lower-leg muscles. 18 patients (8 male,10 female), who had one lower leg (13 right, 5 left) immobilized in a cast, underwent MRI on study days (the nominal day after casting) 3,5,8,15,29 and 43. Both lower legs were imaged simultaneously using a Philips Achieva 3.0T whole body scanner. Cross-sectional area (CSA) was measured for m. tibialis anterior (TA), m gastrocnemius (Gast) and m. soleus (Sol) at 70% of the distance from the lateral malleolus to the tibial tuberosity using a high-resolution T1-weighted spin-echo scan. Total muscle volume was calculated from the product of total CSA and distance from the tibial tuberosity to the insertion of Gas into the Achilles tendon. Pennation angle was measured directly in Gast from a high resolution T1-weighted spin-echo image set angled obliquely. Fractional fat/water ratios were calculated using a 3-point Dixon method from images taken at the 70% position. T2 relaxation times were calculated from 8 spin-echoes with echo times spaced equally between 10 and 80 ms. Values are mean±standard deviation and Student’s t-test was used to assess significance. Total muscle volume reduced by 17% (P<0.001) and the CSA of each muscle by 10.9±6.1% (TA, P=0.001), 21.3±7.4% (GM, P<0.001) and 19.9±9.9% (Sol, P<0.001) in the cast limb, compared with 2%, 6% and 5% in the contralateral limb, over the 6 weeks of immobilization. During immobilization, T2 increased in all 3 muscles: TA 25.9±1.3 ms to 29.0±3.1 ms (P=0.005), Gast 33.8±3.0 ms to 39.3±4.6 ms (P<0.001) and Sol 36.3±2.7 ms to 44.7±5.8 ms (P<0.001). There was no change in fat/water ratio in TA but, unexpectedly in the cast limb given the increase in T2, Gast had 1.4% (P=0.009) and Sol 2.0% (P=0.004) less water than the contralateral limb. Pennation angle reduced from 24.8° to 21.4° in the cast limb, significantly different from the contralateral limb (P=0.013) where no change was seen. Lower leg immobilization for 6 weeks induces measurable changes not only in muscle size but also in parameters reflecting the muscle structure and composition such as muscle fibre pennation angle, T2 relaxation time and fat/water content. Greatest changes in CSA and water content were found in Gast. T2, however, increased most in Sol and, curiously, baseline values were different in all three muscles, possibly reflecting different fibre-type compositions. This study shows that MRI can generate imaging biomarkers of muscle atrophy during immobilization.