HRV (heart rate variability) analysis is a widely employed method to assess cardiac autonomic nervous system activity. Accurate HRV measurement is critical to its value as a diagnostic and prognostic tool. Different breathing patterns may affect HRV, but results obtained under static conditions are conflicting. HRV indices decrease considerably during systemic inflammation evoked by experimental endotoxaemia, enabling the determination of the effects of different breathing patterns on HRV in a dynamic setting. We investigated the impact of different breathing patterns on short-term HRV measurements during experimental endotoxaemia. Furthermore, we assessed whether paced breathing improved HRV reproducibility. Twelve healthy male volunteers received an intravenous bolus (2 ng/kg of body weight) of endotoxin [LPS (lipopolysaccharide), derived from Escherichia coli O:113] on two occasions with an interval of 2 weeks. Five-minute HRV recordings were performed just prior to LPS administration and hourly thereafter until 8 h post-LPS. Three breathing protocols were employed every hour: (i) spontaneous breathing, (ii) metronome-guided breathing at the subject's normal respiratory rate ('paced') and (iii) metronome-guided breathing at 150% of the subject's normal respiratory rate ('mild hyperventilation'). LPS administration resulted in a sharp decrease in all of the HRV indices measured, which was similar during both LPS administrations. Neither paced breathing nor mild hyperventilation influenced HRV indices compared with spontaneous breathing. Paced breathing did not improve reproducibility as it did not exert a significant effect on intra-subject coefficients of variation and intra-class correlation coefficients (calculated between both visits). In conclusion, over a wide range of HRV magnitudes during experimental endotoxaemia, neither paced breathing nor mild hyperventilation affected HRV indices. Moreover, paced breathing did not result in a significant improvement in reproducibility. Therefore employing a paced breathing protocol is not required to obtain valid HRV data during endotoxaemia.