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Patients with chronic fatigue syndrome (CFS) often complain of an inability to maintain activity levels and a variety of autonomic-like symptoms that make everyday activity intolerable at times. The purpose of the study was to determine if there were differences in vagal activity at fixed breathing rates in women with CFS. Twelve women with the diagnosis of CFS between the ages of 32 and 59 years volunteered for the study. Healthy women, who were between the ages of 30 and 49, served as controls. Full signal electrocardiograph and respiratory signals were collected during a paced breathing protocol of three fixed breathing rates (8, 12 and 18 breaths/min) performed in the sitting and standing postures. Vagal activity was analyzed by means of heart rate spectral analysis to determine the subject's response to specific breathing rates and postures. Heart rate variability was used as a non-invasive method of measuring the parasympathetic component of the autonomic nervous system. Using this method, although there was significantly less vagal power in the sitting versus the standing postures for both groups, the overall vagal power was significantly lower (p < 0.034) in the CFS group versus healthy controls. Vagal power was also significantly lower (p < 0.01 to p < 0.05) at all breathing rates in both postures except while standing and breathing at 18 breaths/min. Knowledge of the differences in vagal activity for CFS patients may allow stratification for the analysis of other research variables.