Heart rate variability biofeedback (HRVB) significantly improves heart rate variability (HRV). Breathing at resonance frequency (RF, approximately 6 breaths/min) constitutes a key part of HRVB training and is hypothesized to be a pathway through which biofeedback improves HRV. No studies to date, however, have experimentally examined whether RF breathing impacts measures of HRV. The present study addressed this question by comparing three groups: the RF group breathed at their determined RF for 15 min; the RF + 1 group breathed at 1 breath/min higher than their determined RF for 15 min; and the third group sat quietly for 15 min. After this 15-min period, all groups participated in the Paced Auditory Serial Addition Task (PASAT) for 8 min, and then sat quietly during a 10-min recovery period. HRV, blood pressure, and mood were measured throughout the experiment. Groups were not significantly different on any of the measures at baseline. After the breathing exercise, the RF group reported higher positive mood than the other two groups and a significantly higher LF/HF HRV ratio relative to the control group, a key goal in HRVB training ( p < 0.05). Additionally, the RF group showed lower systolic blood pressure during the PASAT and during the recovery period relative to the control group, with the RF + 1 group not being significantly different from either group ( p < 0.05). Overall, RF breathing appears to play an important role in the positive effect HRVB has on measures of HRV.