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Background: Exercising at intensities where fat oxidation rates are high has been shown to induce metabolic benefits in recreational and health-oriented sportsmen. The exercise intensity (Fat(peak)) eliciting peak fat oxidation rates is therefore of particular interest when aiming to prescribe exercise for the purpose of fat oxidation and related metabolic effects. Although running and walking are feasible and popular among the target population, no reliable protocols are available to assess Fat(peak) as well as its actual velocity (VPFO) during treadmill ergometry. Our purpose was therefore, to assess the reliability and day-to-day variability of VPFO and Fat(peak) during treadmill ergometry running. Conclusion: In summary, relative and absolute reliability indicators for V-PFO and Fat(peak) were found to be excellent. The observed LoA may now serve as a basis for future training prescriptions, although fat oxidation rates at prolonged exercise bouts at this intensity still need to be investigated.