Abstract Following disuse muscle fibres function goes through adaptations such as loss of specific force (Po/CSA) and increase in unloaded shortening velocity (Vo), which could be due to both quantitative changes, i.e. atrophy, and qualitative changes in protein pattern. The underlying mechanisms have not been settled yet. In addition, little is known about the recovery of muscle mass and strength following disuse. Here we report an extensive data set describing in detail functional and protein content adaptations of skeletal muscle in response to both disuse and retraining. Eight young healthy subjects were subjected to 3 weeks unilateral lower limb suspension (ULLS), a widely used human model of disuse skeletal muscle atrophy. Needle biopsies samples were taken from the vastus lateralis muscle pre‐ULLS, post‐ULLS and after 3 weeks recovery during which heavy resistance training was performed (post‐RT). After disuse, cross sectional area (CSA), specific force (Po/CSA) and myosin concentration (MC) decreased both in type 1 and 2A skinned muscle fibres. After recovery, CSA and MC returned to levels comparable to those observed before disuse, whereas Po/CSA and unloading shortening velocity reached a higher level. Myosin heavy chain (MHC) isoform composition of muscle samples did not differ among experimental groups. To study the mechanisms underlying such adaptations, a 2Dproteomic analysis was performed. ULLS induced reduction of myofibrillar, metabolic (glycolytic and oxidative) and antioxidant defense system proteins content. Resistance training was very effective in counteracting ULLS induced alterations indicating that long term ULLS did not prevent the positive effect of exercise on human muscle. This article is protected by copyright. All rights reserved