Lutein (L) and zeaxanthin (Zi) are plant origin carotenoids. They have anti-inflammatory and antioxidant effects. Musclin is a myokine induced by physical activity. The aim of this study was to investigate the effect of L/Zi supplementation on serum and skeletal muscle musclin levels in rats subjected to physical exercise. For this purpose, the rats were divided into four groups, each consisting of seven rats, as the control group, no exercise, L/Zi treated and no exercise group (L/Zi), exercise group (E), and L/Zi treated and exercise group (E + L/Zi treatment). L/Zi was administered daily for eight weeks as an oral supplement by gavage at a dose level of 100 mg/kg BW. When compared with control, E, L/Zi and E + L/Zi decreased muscle triglyceride level by 30.8%, 28.3% and 57.4%, respectively and increased serum and muscle musclin levels by 58.03%, 67.4%, 136.5% and 49.4%, 50.1%, 85.6% respectively. In conclusion, it was determined that skeletal muscle and systemic musclin levels were stimulated by L/Zi combination in rats subjected to chronic exercise.