A circadian pacemaker within the central nervous system regulates the approximately 24-h physiologic rhythms in sleep cycles, hormone secretion, and other physiologic functions. Because the pacemaker cannot be examined directly in humans, markers of pacemaker function must be used to study the pacemaker and its response to environmental stimuli. Core body temperature (CBT), plasma cortisol, and plasma melatonin are three marker variables frequently used to estimate the phase of the human pacemaker. Measurements of circadian phase using markers can contain variability due to the circadian pacemaker itself, the intrinsic variability of the marker relative to the pacemaker, the method of analysis of the marker, and the marker assay. For this report, we compared the mathematical variability of a number of methods of identifying circadian phase from CBT, plasma cortisol, and plasma melatonin data collected in a protocol in which pacemaker variability was minimized using low light levels and regular timing of both the light pattern and the rest/activity schedule. We hoped to assess the relative variabilities of the different physiological markers and the analysis methods. Methods were based on the crossing of an absolute threshold, on the crossing of a relative threshold, or on fitting a curve to all data points. All methods of calculating circadian phase from plasma melatonin data were less variable than those calculated using CBT or cortisol data. The standard deviation for the phase estimates using CBT data was 0.78 h, using cortisol data was 0.65 h, and for the eight analysis methods using melatonin data was 0.23 to 0.35 h. While the variability for these markers might be different for other subject populations and/or less stringent study conditions, assessment of the intrinsic variability of the different calculations of circadian phase can be applied to allow inference of the statistical significance of phase and phase shift calculations, as well as estimation of sample size or statistical power for the number of subjects within an experimental protocol.