Heart rate variability (HRV) is associated with positive physiological and psychological effects. HRV is affected by breathing parameters, yet debate remains regarding the best breathing interventions for strengthening HRV. The objective of the current study was to test whether the inclusion of a postexhalation rest period was effective at increasing HRV, while controlling for breathing rate. A within-subject crossover design was used with 40 participants who were assigned randomly to a breathing pattern including a postexhalation rest period or a breathing pattern that omitted the postexhalation rest period. Participants completed training on each breathing pattern, practiced for 6 min, and sat quietly during a 5-min washout period between practices. Participants were given instructions for diaphragmatic breathing at a pace of six breaths/minute with or without a postexhalation rest period. Recordings of heart rate, breathing rate, HF-HRV, RMSSD, LF-HRV, and SDNN were collected before and during each of the breathing trials. HRV indices were derived from Lead 1 ECG recordings. Pairwise contrasts showed that inclusion of a postexhalation rest period significantly decreased heart rate (p < .001) and increased HF-HRV (p < .05). No differences were found for breathing rates (p > .05), RMSSD (p > .05), and SDNN (p > .05). Results indicated that omission of the postexhalation rest period resulted in higher LF-HRV (p < .05). A postexhalation rest period improves HF-HRV, commonly associated with self-regulatory control, yet the importance of a postexhalation rest period requires further exploration.