Detrended Fluctuation Analysis (DFA) is an algorithm widely used to determine fractal long-range correlations in physiological signals. Its application to heart rate variability (HRV) has proven useful in distinguishing healthy subjects from patients with cardiovascular disease. In this study we examined the effect of respiratory sinus arrhythmia (RSA) on the performance of DFA applied to HRV. Predictions based on a mathematical model were compared with those obtained from a sample of 14 normal subjects at three breathing frequencies: 0.1 Hz, 0.2 Hz and 0.25 Hz. Results revealed that: (1) the periodical properties of RSA produce a change of the correlation exponent in HRV at a scale corresponding to the respiratory period, (2) the short-term DFA exponent is significantly reduced when breathing frequency rises from 0.1 Hz to 0.2 Hz. These findings raise important methodological questions regarding the application of fractal measures to short-term HRV.