Short successive periods of muscle disuse, due to injury or illness, can contribute significantly to the loss of muscle mass with aging (sarcopenia). It has been suggested that increasing the protein content of the diet may be an effective dietary strategy to attenuate muscle disuse atrophy. We hypothesized that protein supplementation twice daily would preserve muscle mass during a short period of limb immobilization. Twenty-three healthy older (69 ± 1 y) men were subjected to 5 d of one-legged knee immobilization by means of a full-leg cast with (PRO group; n = 11) or without (CON group; n = 12) administration of a dietary protein supplement (20.7 g of protein, 9.3 g of carbohydrate, and 3.0 g of fat) twice daily. Two d prior to and immediately after the immobilization period, single-slice computed tomography scans of the quadriceps and single-leg 1 repetition maximum strength tests were performed to assess muscle cross-sectional area (CSA) and leg muscle strength, respectively. Additionally, muscle biopsies were collected to assess muscle fiber characteristics as well as mRNA and protein expression of selected genes. Immobilization decreased quadriceps' CSAs by 1.5 ± 0.7% (P < 0.05) and 2.0 ± 0.6% (P < 0.05), and muscle strength by 8.3 ± 3.3% (P < 0.05) and 9.3 ± 1.6% (P < 0.05) in the CON and PRO groups, respectively, without differences between groups. Skeletal muscle myostatin, myogenin, and muscle RING-finger protein-1 (MuRF1) mRNA expression increased following immobilization in both groups (P < 0.05), whereas muscle atrophy F-box/atrogen-1 (MAFBx) mRNA expression increased in the PRO group only (P < 0.05). In conclusion, dietary protein supplementation (∼20 g twice daily) does not attenuate muscle loss during short-term muscle disuse in healthy older men. This trial was registered at clinicaltrials.gov as NCT01588808.