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. Abstract The object of the study was to investigate the sequential changes of protein synthesis in skeletal muscle during establishment of obesity, considering muscle typology. Adult Wistar rats were fed a standard diet for 16 weeks (C; n= 14), or a high-fat, high-sucrose diet for 16 (HF16; n= 14) or 24 weeks (HF24; n= 15). Body composition was measured using a dual-energy X-ray absorptiometry scanner. The fractional synthesis rates (FSRs) of muscle protein fractions were calculated in tibialis anterior (TA) and soleus muscles by incorporation of l-13C-valine in muscle protein. Muscle lipid and mitochondria contents were determined using histochemical analysis. Obesity occurred in an initial phase, from 1 to 16 weeks, with an increase in weight (P < 0.05), fat mass (P < 0.001), muscle mass (P < 0.001) and FSR in TA (actin: 5.3 +/- 0.2 vs. 8.8 +/- 0.5% day-1, C vs. HF16, P < 0.001) compared with standard diet. The second phase, from 16 to 24 weeks, was associated with a weight stabilization, a decrease in muscle mass (P < 0.05) and a decrease in FSR in TA (mitochondrial: 5.6 +/- 0.2 vs. 4.2 +/- 0.4% day-1, HF16 vs. HF24, P < 0.01) compared with HF16 group. Muscle lipid content was increased in TA in the second phase of obesity development (P < 0.001). Muscle mass, lipid infiltration and muscle protein synthesis were differently affected, depending on the stage of obesity development and muscle typology. Chronic lipid infiltration in glycolytic muscle is concomitant with a reduction of muscle protein synthesis, suggesting that muscle lipid infiltration in response to a high-fat diet is deleterious for the incorporation of amino acid in skeletal muscle proteins.