Heart rate variations reflect the output of the complex control of the heart mediated by the autonomic nervous system. Because of that, they also encode different types of information, namely the efferent outflow of reflex mechanisms involved in the beat-to-beat control of cardiac function, the efferent activity of neurohumoral elements involved in the control of other cardiovascular parameters and random noise resulting from the hysteresis of the different controllers. The degree to which power spectrum estimation methods will uncover the periodic component of heart rate variations is in direct relation with the status of the system under study. Although the utility of spectral methods is now established in mammalian research, very little is known on the utility of these techniques in non-mammalian cardiovascular research. This review covers this space by discussing the physiological significance of heart rate variations in non-mammalian vertebrates. A detailed account of the different steps of the technique, its limitations and the ways to overcome these problems are also presented. These are: the recording of the cardiac event signal, the detection and digital processing methods, the satisfaction of stationarity conditions, the problem of spectral leakage and the different methods to estimate the power spectrum.