Heart rate variability in the 0.1 Hz frequency range has been reported to provide a non-invasive measure of sympathetic activity, however the technique has not been validated for the quantification of differences in sympathetic activity between individuals. We compared 0.1 Hz power with cardiac noradrenaline spillover and muscle sympathetic nerve activity in conditions of sympathetic denervation, including pure autonomic failure and after cardiac transplantation, and in the context of sympathetic nervous activation, in cardiac failure and in relation to ageing and during application of mental stress and isometric exercise. While there was agreement between techniques in pure autonomic failure and early after transplantation, disparity between spectral analysis and the more direct indices of sympathetic function were obtained for cardiac failure and ageing. Among healthy subjects, in whom measurements were made simultaneously, there was no significant correlation between 0.1 Hz power in either absolute units or normalised for total power and the rate of noradrenaline spillover from the heart to plasma. Heart rate variability clearly provides a functional measure of cardiac sympathetic mechanisms which is dependent on post-synaptic aspects of neural function, in addition to rates of sympathetic nerve firing. One particular application of heart rate spectral analysis in neurophysiological research might be in combination with the cardiac noradrenaline spillover technique, allowing more comprehensive assessment of both neuronal and post-synaptic aspects of the cardiac neuroeffector response.