Heart rate variability (HRV) analysis is a powerful instrument that provides information about the heart conditions. However, there exist some limitations in the use of HRV in the clinical practice. Examples are the lack of reference values for healthy populations, different HR (Heart Rate) acquisition systems, and varying software packages. Other factors that affect HRV values are the influence of lifestyle, drugs and alcohol consumption, and pollution. In this work, recommendations to perform HRV-based experiments were established. These suggestions refer to best moment of the day to record data, the optimal body position, and the quality and duration of the recorded signals. In this way, HR data from 6 healthy subjects (2 women, 4 men), with median age of 50 years old, were recorded during 15 days, 3 times a day. Recordings were performed in the following situations: both supine and sitting body positions, in the morning, in the afternoon and at night. Data were processed and HRV analysis was performed. Distorting factors affecting HRV have been determined. The most stable HRV indexes (less variation over the days) have also been established. For this task, a variation coefficient was calculated for each parameter, as the ratio between the standard deviation and the mean value. Results indicated that HR data should be recorded in the morning, the sitting position. Related to signals duration, when comparing HR signals, they should be of equal length (same recording time). In addition, HRVi (HRV triangular index) and MADRR (median of the absolute differences between adjacent RR intervals) resulted in the most robust indexes in both low and high frequency domains. For global indexes, the ApEn (approximate entropy) measure emerged as the most stable one. As a conclusion, researchers must be extremely cautious in studies involving HRV analysis; the moment of the day to record data, the body position, or the quality of recorded data will produce different HR signals, and thus, the values of the HRV parameters will be different in each case. This may clearly bias the conclusions of the study.