The associations between cardiovascular disease (CVD) risk factors and heart rate variability (HRV) have shown some inconsistencies. To examine the impact of the different Kubios threshold-based artefact correction levels on the associations between different CVD risk factors and a heart rate variability (HRV) score in three independent human cohorts. A total of 107 children with overweight/obesity, 132 young adults, and 73 middle-aged adults were included in the present study. Waist circumference and the HRV score were negatively associated using the medium and the strong Kubios filters in children (beta = -0.22 and - 0.24, P = 0.03 and 0.02 respectively) and the very strong Kubios filter in middle-aged adults (beta = -0.39, P = 0.01). HDL-C was positively associated with the HRV score across Kubios filters (beta ranged from 0.21 to 0.31, all P </= 0.04), while triglycerides were negatively associated with the HRV score using the very strong Kubios filter in young adults (beta = -0.22, P = 0.02). Glucose metabolism markers (glucose, insulin, and HOMA index) were inversely associated with the HRV score across Kubios filters in young adults (beta ranged from -0.29 to -0.22; all P </= 0.03). Importantly, most of these associations disappeared after including HR as a covariate, especially in children and young adults. It should be mandatory to report the Kubios filter used and to include the HR (as a confounder factor) to allow the comparability of the results across different studies.