Low values on heart rate variability (HRV) derived parameters at resting have been used to predict cardiovascular diseases (CVD) and mortality. In this regard, short-term HRV recordings (usually from 5 to 15 minutes) are increasing their popularity because data acquisition can be performed under more controlled conditions than long-term recordings (e.g., 24 hours). However, different methodological aspects before, during, and after the HRV assessment could affect the quantification and the clinical interpretations of the HRV-derived parameters, as well as hamper comparisons across different studies. In the present study, we summarize these methodological aspects that should be considered in both the research and the clinical settings. These are: (1) the validity and reproducibility of the device used to assess the HRV; (2) the influence of the software used to perform the artefact correction; (3) previous conditions before the testing day; (4) establishing the proper conditions during the HRV assessment (e.g., controlled respiratory frequency); (5) after assessing the HRV, considering the "best" data selection and statistical analysis approach; and (6) the role of the heart rate on the associations between the different CVD risk factors outcomes (e.g., cardiorespiratory fitness) and the HRV-derived parameters.