Aim The impact of disuse on the loss of skeletal muscle mass and strength has been well documented. Given that most studies have investigated muscle atrophy after more than 2 weeks of disuse, few data are available on the impact of shorter periods of disuse. We assessed the impact of 5 and 14 days of disuse on skeletal muscle mass, strength and associated intramuscular molecular signalling responses. Methods Twenty‐four healthy, young (23 ± 1 year) males were subjected to either 5 (n = 12) or 14 (n = 12) days of one‐legged knee immobilization using a full leg cast. Before and immediately after the immobilization period, quadriceps muscle cross‐sectional area (CSA), leg lean mass and muscle strength were assessed, and biopsies were collected from the vastus lateralis. Results Quadriceps muscle CSA declined from baseline by 3.5 ± 0.5 (P < 0.0001) and 8.4 ± 2.8% (P < 0.001), leg lean mass was reduced by 1.4 ± 0.7 (P = 0.07) and 3.1 ± 0.7% (P < 0.01) and strength was decreased by 9.0 ± 2.3 (P < 0.0001) and 22.9 ± 2.6% (P < 0.001) following 5 and 14 days of immobilization respectively. Muscle myostatin mRNA expression doubled following immobilization (P < 0.05) in both groups, while the myostatin precursor isoform protein content decreased after 14 days only (P < 0.05). Muscle MAFBx mRNA expression increased from baseline by a similar magnitude following either 5 or 14 days of disuse, whereas MuRF1 mRNA expression had increased significantly only after 5 days. Conclusion We conclude that even short periods of muscle disuse can cause substantial loss of skeletal muscle mass and strength and are accompanied by an early catabolic molecular signalling response.