Background Disuse of a muscle group, which occurs during bedrest, spaceflight, and limb immobilization, results in atrophy. It is unclear, however, if the magnitude of decline in skeletal muscle quality is similar to that for muscle size. The purpose of this study was to examine the effects of two weeks of knee joint immobilization on vastus lateralis and rectus femoris echo intensity and cross-sectional area. Methods Thirteen females (mean ± SD age = 21 ± 2 years) underwent two weeks of left knee joint immobilization via ambulating on crutches and use of a brace. B-mode ultrasonography was utilized to obtain transverse plane images of the immobilized and control vastus lateralis and rectus femoris at pretest and following immobilization. Effect size statistics and two-way repeated measures analyses of variance were used to interpret the data. Results No meaningful changes were demonstrated for the control limb and the rectus femoris of the immobilized limb. Analyses showed a large increase in vastus lateralis echo intensity (i.e., decreased muscle quality) for the immobilized limb (p = .006, Cohen’s d = .918). For vastus lateralis cross-sectional area, no time × limb interaction was observed (p = .103), but the effect size was moderate (d = .570). There was a significant association between the increase in vastus lateralis echo intensity and the decrease in cross-sectional area (r = − .649, p = .016). Conclusion In female participants, two weeks of knee joint immobilization resulted in greater deterioration of muscle quality than muscle size. Echo intensity appears to be an attractive clinical tool for monitoring muscle quality during disuse.