Heart Rate Variability (HRV) has been extensively investigated for characterizing the autonomic nervous system (ANS) in controlling heart rate. Since ectopic beats, artefacts and noise of the ECG can affect the estimation of HRV features, pre-processing of the RR tachogram can improve the accuracy of HRV analysis and discriminatory power. This paper investigates the effect of different automated preprocessing methods on discriminatory capability of HRV analysis with an example of comparison between different groups of normal and type II diabetic patients with different Angiotensin-Converting Enzyme (ACE) gene polymorphism. Results show that smaller p-values and therefore higher discriminatory capability are found when preprocessing is used, while none of the features can show significant difference if they are estimated from the raw R-R sequence. Secondly, the preprocessing methods do not have the same effect for all HRV features.