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The aim of this study was to investigate the rate of Myostatin (MSTN) and Follistatin (FLST) gene expression in fast and slow twitch muscles in response to 8 weeks of high intensity interval training (HIIT).  To this end, 12 male 8-week old Wistar rats were randomly classified into two groups of exercise (n=6) and control (n=6). Exercise group performed a progressive HIIT protocol, five times weekly for 8 weeks using alternating sprint running for 30 min/session that divided into three 4min bouts (35-50m/min speed, >90% VO2 max) separated by a 2min active recovery period (30-50% VO2 max). Gene expression levels for MSTN and FLST in Soleus (SOL) and Extensor digitorum longus (EDL) muscles were measured by real-time RT-PCR. MSTN mRNA levels has slightly (7%); yet significantly (p<0.001), decreased in SOL muscle, while expression of this gene in EDL muscle decreased (30%) (p<0.001). Moreover, FLST gene expression in SOL muscle increased 31% (p<0.001). Interestingly, level of FLST gene expression in EDL muscle increased 100% compared to control group (p<0.001).The results provide a perspective regarding muscle differentiation in MSTN and FLST and their variation in response to HIIT and suggests that HIIT may be an effective way to decrease adipose tissue by MSTN and FLST regulation in skeletal muscles.