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 both the constant routine and daily routine protocols, during which HRV and CBT were continuously monitored. During the constant routine, the subjects were kept awake for 27 h in a semi-recumbent posture with minimal physical activity; small isocaloric meals were provided every 2 h. During the daily routine, the subjects carried on their lives normally. Data were analyzed using generic spectral analysis (GSA) based on a fast Fourier transform; coarse graining spectral analysis (CGSA) 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 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) CGSA eliminated diet-induced peaks in GSA-based HRV spectra during the constant routine and emphasized the crossover at 10^-3.5 Hz; and 4) CBT correction did not alter the results. Below a frequency of 10^-3.5Hz (a period longer than 1 h), HRV is strongly influenced by behavioral factors; above that crossover frequency, HRV is behavior-independent, possibly reflecting an intrinsic regulatory system.