The effects of such behavioral factors as physical activity, food intake, and circadian rhythm on long-term heart rate variability (HRV) in humans remain poorly understood. We therefore studied their effects on HRV using a constant-routine protocol that included simultaneous core body temperature (CBT) correction. Seven healthy subjects completed the constant-routine and daily-routine protocols, during which HRV and CBT were continuously monitored. During the constant routine, subjects were kept awake for 27 h in a semirecumbent posture with minimal physical activity; small isocaloric meals were provided every 2 h. During the daily routine, subjects carried on their lives normally. Data were analyzed using generic spectral analysis based on a fast Fourier transform; coarse-graining spectral analysis was also used to eliminate periodicity due to the regular meals for raw HRV and for the CBT-corrected HRV without circadian and/or low-frequency ultradian components. The results showed that 1) the power spectra of HRV in the constant routine and daily routine had similar power-law scalings at frequencies above approximately 10(-3.5) Hz, while 2) below that crossover frequency, HRV was smaller in the constant routine than in the daily routine, with the difference becoming significant (P < 0.05) at <10(-4) Hz, 3) coarse-graining spectral analysis eliminated diet-induced peaks in generic spectral analysis-based HRV spectra during the constant routine and emphasized the crossover at approximately 10(-3.5) Hz, and 4) CBT correction did not alter the results. Below a frequency of approximately 10(-3.5) Hz (a period >1 h), HRV is strongly influenced by behavioral factors; above that crossover frequency, HRV is behavior independent, possibly reflecting an intrinsic regulatory system.