BACKGROUND AND AIMS: Resting heart rate variability (HRV) and maximal fat oxidation (MFO) during exercise are both considered as a noninvasive biomarkers for early detection of cardiovascular risk factors. Thus, this study aimed to analyze the relationship between resting HRV parameters and MFO during exercise, and the intensity of exercise that elicit MFO (Fat(max)) in healthy sedentary adults. METHODS AND RESULTS: A total of 103 healthy young adults (22.2 +/- 2.3 years old, 67% female; from the ACTIBATE cohort) and 67 healthy middle-aged adults (53.1 +/- 5.0 years old, 52% female; from the FIT-AGEING cohort) were included in this cross-sectional study. HRV was assessed using a Polar RS800CX heart rate monitor, while MFO and Fat(max) were determined during a graded exercise treadmill test using indirect calorimetry. No significant associations were observed for healthy young adults (standardized beta coefficients ranged from -0.063 to 0.094, and all P >/= 0.347) and for middle-aged adults (standardized beta coefficients ranged from -0.234 to 0.090, and all P >/= 0.056). Nevertheless, only a weak association was observed between one HRV parameter in time-domain (the percentage of R-R intervals that shows a difference higher than 50 ms [pNN50]) and MFO in the cohort of middle-aged adults (beta coefficient = -0.279, and P = 0.033). CONCLUSION: The results of this study suggest that resting HRV parameters are not associated with MFO and Fat(max) during exercise in two independent cohorts of healthy sedentary young and middle-aged adults, respectively.