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Exploring the independent effect of mechanical discharge in response to weight loss (WL) seems necessary but remains highly challenging from a methodological point. Anti-gravity treadmills could be relevant to simulate a mechanical WL by body weight support (BWS), but their reliability remains unclear to properly explore exercise energy metabolism, especially at low degrees of alleviations. The study aimed to evaluate the accuracy and reproducibility of an anti-gravity treadmill to generate BWS, and the reproducibility of cardiometabolic responses to an exercise performed at low degrees of BWS on this device. Observed BWS of 18 normal-weight males was obtained twice at seven degrees of target BWS (i.e., 0, 3, 6, 9, 12, 15, 18%) using a digital scale inside the anti-gravity treadmill, and was compared to the expected BWS. Then, 15 of them performed 5-min bout of low-intensity walking exercise at these degrees of BWS in a randomized order, separated by 4-min rest. The exercise was identically repeated on three occasions separated by a minimum of 3 days. Energy metabolism and heart rate (HR) were measured throughout the exercise by indirect calorimetry and a HR monitor, respectively. The observed BWS were significantly different from the expected BWS (p< 0.001), and there was a high inter- and intra-individual variability in BWS generated by the anti-gravity treadmill. Results showed an overall good reliability of VO2 (intraclass correlation coefficients (ICC) values ranging from 0.67 to 0.85) and HR (ICC > 0.8) in response to exercise. An effect of the degree of BWS was observed for VO2 (p< 0.001), illustrating reduced values at 15% and 18% of BWS compared to 0, 3, and 6%. Such device might not be adapted to simulate low degrees of WL in normal-weight males, particularly when it comes to the exploration of energy metabolism. [ABSTRACT FROM AUTHOR]