Background: Higher baroreflex sensitivity, enhanced high frequency component of heart rate variability and a faster heart rate recovery with cessation of exercise in elite athletes suggests plasticity in the central nervous mechanisms that control the heart. This experimental study was designed to directly test the hypothesis that the strength of parasympathetic tone determines exercise capacity. We hypothesised that vagal withdrawal should decrease and vagal recruitment should enhance exercise capacity. We targeted vagal preganglionic neurones of the dorsal motor nucleus of the vagus nerve (DVMN) which provide functional innervation of the left cardiac ventricle (Machhada et al., 2015; 2016). Methods: In male Sprague-Dawley rats (380-420 g), DVMN neurones were transduced to express an inhibitory Gi-protein-coupled Drosophila allatostatin receptor (AlstR) (n=8) or green fluorescent protein (GFP) as a control (n=8). Application of the insect peptide ligand allatostatin (5 µl) produces rapid, selective inhibition of targeted neurones. A pharmacological study investigated the role of muscarinic and neuronal nitric oxide-mediated mechanisms using systemic treatment with atropine methyl nitrate (2 mg/kg, i.p., n=5) or selective neuronal NO synthase inhibitor 7-nitroindazole (7-NI) (30 mg/kg, i.p., n=8). For optogenetic activation, DVMN neurones were targeted to express an optogenetic construct ChiEF (n=9) or control transgene GFP (n=10) and stimulated with blue laser light (445 nm, 10 ms pulses, 15 Hz, 15 min). Exercise capacity was determined using a treadmill with a shock grid set at the minimum of 0.1 mA. Rats were preselected for their compliance after a three day recruitment protocol and randomized. The experimental protocol involved starting speeds of 20-30 cm/s over 5 min after 15 min acclimatisation. Speeds were then raised in 5 cm/s increments every 5 min until the hind limbs made grid contact four times within a 2 min period. The calculated work (Joules, J) was used as an index of exercise capacity. Results: Acute inhibition of the DVMN neurones by allatostatin resulted in a dramatic reduction in exercise capacity (8±2 vs 202±27 J; p<0.0001; ANOVA). In rats given atropine and vehicle no significant differences in exercise capacity were observed (113±20 vs 112±22 J, p=0.9; t-test). Systemic administration of 7-NI was associated with a significant reduction in exercise capacity (33±19 vs 129±19 J, p=0.0002; t-test), as did 4 h of atropine treatment (63±12 vs 116±20 J, p=0.0019; t-test). Rats expressing ChIEF by the DVMN neurones displayed a significantly higher exercise capacity 4 days following optogenetic stimulation (94±11 vs 47±6 J; p=0.002; ANOVA). Improvements were similar to that observed in naïve rats trained to exhaustion over the same period (105±16 vs 47±6 J in rats expressing GFP; p<0.0001; ANOVA). Conclusion: These results suggest that the strength of parasympathetic tone determines exercise capacity.