Purpose Strength-related exerkines may be promoted in response to exercise imposing high mechanical stress on the skeletal muscles. For a constant velocity, the inclination of the slope during downhill exercise influences the total mechanical load. The aim of the study was to compare exerkines release following two prolonged decline walking (-10% and -20%) exercise session. Methods Twelve moderately active healthy men (age: 23.3 ± 3.6 years; mass: 70.0 ± 4.5 kg; height: 177.2 ± 2.9 cm; body fat: 13.1 ± 3.2%) performed two 45-min downhill treadmill exercises at -10% (DOWN10) and -20% (DOWN20) slopes at the same pace (6 km.h−1). Heart rate and muscle oxygenation were monitored during exercise and blood samples were taken before, 1 h and 24 h after exercise. Results Heart rate and muscle oxygenation were respectively greater and lower during DOWN20 exercise than DOWN10. Myostatin (MSTN; DOWN10 p ≤ 0.01 and DOWN20 p ≤ 0.05) and interleukine-6 (DOWN10 and DOWN20 p ≤ 0.001) levels increased 1 h-post exercise in both conditions while no difference was found for decorin, follistatin and the growth differentiation factor-15 levels. No difference on exerkines release was evidenced between − 10% and − 20% treadmill slope but a significant condition effect between DOWN20 and DOWN10 was observed for DCN (p ≤ 0.05). Conclusion A 45-min downhill walking exercise at a − 10% slope represents a valid strategy to stimulate the cardiovascular and muscular functions, as inferred from the increase in MSTN and IL-6 exerkines levels, and the use of a steepest slope protocol is no a prerequisite to elicit greater responses.