The objective assessment of pain is difficult in animals and humans alike. Detrended fluctuation analysis (DFA) is a method which extracts "hidden" information from heart rate time series, and may offer a novel way of assessing the subjective experience associated with pain. The aim of this study was to investigate whether any fractal differences could be detected in heart rate time series of sheep due to the infliction of ischaemic pain. Heart rate variability (HRV) was recorded continuously in five ewes during treatment sequences of baseline, intervention and post-intervention for up to 60 min. Heart rate time series were subjected to a DFA, and the median of the scaling coefficients (alpha) was found to be alpha=1.10 for the baseline sequences, 1.01 for the intervention sequences and 1.00 for the post-intervention sequences. The complexity in the regulation of heartbeats decreased between baseline and intervention (p approximately 0.03) and baseline and post-intervention (p approximately 0.01), indicating reperfusion pain and nociceptive sensitization in the post-intervention sequence. Random time series based on Gaussian white noise were generated, with similar mean and variance to the HRV sequences. No difference was found between these series (p approximately 0.28), pointing to a true difference in complexity in the original data. We found no difference in the scaling coefficient alpha between the different treatments, possibly due to the small sample size or a fear induced sympathetic arousal during test day 1 confounding the results. The decrease in the scaling coefficient alpha may be due to sympathetic activation and vagal withdrawal. DFA of heart rate time series may be a useful method to evaluate the progressive shift of cardiac regulation toward sympathetic activation and vagal withdrawal produced by pain or negative emotional responses such as fear.