Heart rate variability (HRV), the beat-to-beat alterations in heart rate, comprises sympathetic and parasympathetic nerve activities of the heart. HRV analysis is used to quantify cardiac autonomic regulation. Since respiration could be a confounding factor in HRV evaluation, some studies recommend consciously controlled breathing to standardize the method. However, it remains unclear whether controlled breathing affects HRV measurement. We compared the effects of controlled breathing on HRV with those of spontaneous breathing. In 20 healthy volunteers, we measured respiratory frequency (f), tidal volume, and blood pressure (BP) and recorded electrocardiograms during spontaneous breathing (14.8 ± 0.7 breaths/min) and controlled breathing at 15 (0.25 Hz) and 6 (0.10 Hz) breaths/min. Compared to spontaneous breathing, controlled breathing at 0.25 Hz showed a higher heart rate and a lower high-frequency (HF) component, an index of parasympathetic nerve activity, although the f was the same. During controlled breathing at 0.10 Hz, the ratio of the low frequency (LF) to HF components (LF/HF), an index of sympathetic nerve activity, increased greatly and HF decreased, while heart rate and BP remained almost unchanged. Thus, controlled breathing at 0.25 Hz, which requires mental concentration, might inhibit parasympathetic nerve activity. During controlled breathing at 0.10 Hz, LF/HF increases because some HF subcomponents are synchronized with f and probably move into the LF band. This increment leads to misinterpretation of the true autonomic nervous regulation. We recommend that the respiratory pattern of participants should be evaluated before spectral HRV analysis to correctly understand changes in autonomic nervous regulation.