The aim of this study is to examine heart rate variability (HRV) alteration after the injection of atropine and saline and to determine if ensemble averaging affects the outcome of frequency domain analysis. The HRV data is pre-processed using ensemble averaging and then frequency domain and statistical analysis is done. The HRV readings originate from a previous study of four rats. Ensemble averaging reduces the mean drift effect in the data. The original and ensemble is then processed through an Fast Fourier Transform (FFT). The results show that the ensembled data allows frequency peaks and changes to be seen more clearly than the FFT of the original data set. Through statistical analysis it is also shown that the change in HRV from the atropine injection is much greater than the change in HRV from the saline injection.