The purpose of this study was to determine if muscle activity of the biceps followed by isometric flexion changes T₂ measured in the biceps. It is hypothesized that an increase in T₂ will be observed in the biceps but not in the triceps after flexion exercise. Ten healthy volunteers were imaged with a one-channel neck coil while seated in a 0.5 T upright open magnetic resonance imaging (MRI) scanner using a three-dimensional double echo steady-state (DESS) sequence. Volunteers were imaged while relaxing their arm for 10, 20, and 30  min during an isometric biceps flexion immediately following performance of biceps curls to exhaustion, and again after relaxing for 10 and 20 min. Voxel-wise T₂ was calculated by fitting to a DESS signal equation in regions segmented at muscle centers to determine mean T₂. During isometric biceps flexion immediately following biceps curls, mean T₂ increased (average 33%, p < 0.05) in the biceps but not in the triceps. By 20 min after curls, mean T₂ decreased (p < 0.05), and was near preactivity values. In contrast, there was no change in triceps T₂ across any activity or postactivity time points. Intra-rater repeatability was excellent (ICC:​ 0.90–0.97). This study demonstrated that measuring T₂ in an active muscle is feasible using a DESS sequence in an upright open MRI scanner. This could enable the study of muscle function while the muscle is working and weight-bearing, rather than of the “fatigue” of the muscles after activity. In comparison to electromyography, MRI also enables the study of deep muscles and allows simultaneous assessment of activity and function.