Key points • Prolonged obesity leads to ectopic lipid accumulation in non-adipose tissues, particularly in skeletal muscles, inducing metabolic dysfunctions (reduced glucose uptake, mitochondria dysfunction, lipotoxicity). • Several studies in humans and rodents have shown that obesity induces a short-term increase in fat-free mass but a long-term decrease in skeletal muscle mass. • We investigated the mechanisms potentially involved in muscle loss by measuring simultaneously protein synthesis and lipid infiltration in different types of skeletal muscles, during the development of obesity. • Our results show that protein synthesis rate in glycolytic muscles increased together with muscle mass during the early phase of obesity development, whereas it decreased later. Reduced protein synthesis rate was associated with a high lipid accumulation in glycolytic muscles. • These results suggest that lipid accumulation in muscles during prolonged obesity is deleterious for amino acid incorporation in skeletal muscle proteins, and thus indirectly for muscle mass.