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Heart rate variability (HRV) is a major noninvasive technique for evaluating the autonomic nervous system (ANS). Use of time-frequency approach to analyze HRV allows investigating the ANS behavior from the power integrals, as a function of time, in both steady-state and non steady-state. Power integrals are examined mainly in the low-frequency and the high-frequency bands. Traditionally, constant boundaries are chosen to determine the frequency bands of interest. However, these ranges are individual, and can be strongly affected by physiologic conditions (body position, breathing frequency). In order to determine the dynamic boundaries of the frequency bands more accurately, especially during autonomic challenges, we developed an algorithm for the detection of individual time-dependent spectral boundaries (ITSB). The ITSB was tested on recordings from a series of standard autonomic maneuvers with rest periods between them, and the response to stand was compared to the known physiological response. A major advantage of the ITSB is the ability to reliably define the mid-frequency range, which provides the potential to investigate the physiologic importance of this range.