Heart rate variability and post-exercise heart rate recovery are used to assess cardiac parasympathetic tone in human studies, but in some cases these indices appear to yield discordant information. We utilized pyridostigmine, an acetylcholinesterase inhibitor that selectively augments the parasympathetic efferent signal, to further characterize parasympathetic regulation of rest and post-exercise heart rate. We measured time-domain and frequency-domain indices of resting heart rate variability and post-exercise heart rate recovery in 10 sedentary adults and 10 aerobically-trained athletes after a single oral dose of pyridostigmine 30 mg and matching placebo in randomized, double-blind, crossover trial. In sedentary adults, pyridostigmine decreased resting heart rate (from 66.7 (12.6) beats/min to 58.1 (7.6) beats/min, p = 0.005 vs. placebo) and increased post-exercise heart rate recovery at one minute (from 40.7 (10.9) beats/min to 45.1 (8.8) beats/min, p = 0.02 vs. placebo). In trained athletes, pyridostigmine did not change resting heart rate or post-exercise heart rate recovery when compared with placebo. Time-domain and frequency-domain indices of resting heart rate variability did not differ after pyridostigmine vs. placebo in either cohort and were not significantly associated with post-exercise heart rate recovery in either cohort. The divergent effects of pyridostigmine on resting and post-exercise measures of cardiac parasympathetic function in sedentary subjects confirm that these measures characterize distinct aspects of cardiac parasympathetic regulation. The lesser effect of pyridostigmine on either measure of cardiac parasympathetic tone in the trained athletes indicates the enhanced parasympathetic tone associated with exercise training is at least partially attributable to adaptations in the efferent parasympathetic pathway.