Patients with severe traumatic brain injury (TBI) may have autonomic dysfunction, one manifestation of which is orthostatic intolerance. This potentially impairs physical rehabilitation. However, the exact mechanisms remain elusive. In 30 patients participating in a trial of early tilt training versus standard care and 15 healthy volunteers, 5-min electrocardiography was recorded in the supine position and during 70° head-up tilt. Heart rate variability was analyzed by the low- and high-frequency (LF and HF) power, the LF-HF ratio, the total power, the ratio of the standard deviation of normal-to-normal intervals (SDNN), the root mean square of successive differences (RMSSD), the detrended fluctuations, and sample entropy. In patients in the upright compared to the supine position, SDNN (p < 0.001), RMSSD (p < 0.001), and total power (p = 0.004) all decreased, while the remaining variables were unchanged; no long-term differences in heart rate variability in the supine position were found between early tilt training and standard care. In the healthy volunteers, all measures besides SDNN and total power changed significantly between supine and upright position. In patients with severe TBI compared to healthy volunteers, several measures of heart rate variability changed differentially during mobilization from the supine to the upright position.