Introduction: Respiratory sinus arrhythmia (RSA), a main component of heart rate variability (HRV), maximises at a breathing frequency (FR) of 4-6 b.min-1 (1), and correlates with cardiac vagal activity (2). The independent influences of FR and concomitant changes in tidal volume (VT) and PCO2 upon HRV and autonomic nervous system (ANS) function are unknown. We tested the hypothesis that HRV may be maximised by controlling both FR and VT.Methods: Nine healthy, recreationally active men participated (27.1±2.6 yr). In part 1 the relationship between FR was assessed and individual FR optima identified, whilst maintaining a constant VT of 30% of vital capacity (VC). In part 2 the optimal FR identified in part 1 was implemented across a range of VT. Parts 1 and 2 included a semi-spontaneous condition in which only VT or FR were controlled, respectively. Conditions were randomised and made in an upright-reclined position. Mild hypercapnia was maintained under all conditions except semi-spontaneous breathing (GA-200 gas analyser, iWorx Systems Inc.). A biofeedback system (LabView, National Instruments Inc.) specified respiratory flow and duty cycle, which were measured by heated pneumotachograph (Hans Rudolph 3813, Hans Rudolph Inc.), whilst heart rate was measured via 3-lead ECG. Primary outcomes were the standard deviation of normal R-R intervals (SDNN) and the total power (TP) of the power spectrum density. Repeated measures ANOVA with post hoc pairwise comparisons using Bonferroni correction were used to test differences between conditions and Pearson correlations accessed the inter-relationships.Results:In 3 of 9 participants SDNN and TP maximised at 4 b.min-1, whilst the remaining 6 maximised at 6 b.min-1. Significant main effects of FR and VT were found for SDNN [F (5, 40) = 8.195; p<0.001; F (6, 48) = 13.280; p<0.001] and TP [F (5, 40) = 11.147; p<0.001; F (6, 48) = 7.233; p<0.001] respectively. Effect sizes were moderate for all variables and conditions (ηp2=0.475-0.624). Figures 1 A-D summarise the mean ± S.E.M. for all conditions.Discussion: There were significant independent effects of FR and VT, upon SDNN and TP, with maxima at 6 b.min-1 for 6/9 participants, and at the highest VT for all participants. Adopting a higher VT than that used spontaneously at optimal FR did not significantly increase HRV. Whether VTs over 40% VC yield significantly greater HRV remains to be explored.