We elucidated the autonomic mechanisms whereby heart rate (HR) is regulated by the muscle metaboreflex. Eight male participants (22 ± 3 years) performed three exercise protocols: (1) enhanced metaboreflex activation with partial flow restriction (bi-lateral thigh cuff inflation) during leg cycling exercise, (2) isolated muscle metaboreflex activation (post-exercise ischaemia; PEI) following leg cycling exercise, (3) isometric handgrip followed by PEI. Trials were undertaken under control (no drug), β1-adrenergic blockade (metoprolol) and parasympathetic blockade (glycopyrrolate) conditions. HR increased with partial flow restriction during leg cycling in the control condition (11 ± 2 beats min(-1); P < 0.05). The magnitude of this increase in HR was similar with parasympathetic blockade (11 ± 2 beats min(-1)), but attenuated with β-adrenergic blockade (4 ± 1 beats min(-1); P < 0.05 vs. control and parasympathetic blockade). During PEI following leg cycling exercise, HR remained similarly elevated above rest under all conditions (11 ± 2, 13 ± 3 and 9 ± 4 beats min(-1), for control, β-adrenergic and parasympathetic blockade; P > 0.05 between conditions). During PEI following handgrip, HR was similarly elevated from rest under control and parasympathetic blockade (4 ± 1 vs. 4 ± 2 beats min(-1); P > 0.05 between conditions) conditions, but attenuated with β-adrenergic blockade (0.2 ± 1 beats min(-1); P > 0.05 vs. rest). Thus muscle metaboreflex activation-mediated increases in HR are principally attributable to increased cardiac sympathetic activity, and only following exercise with a large muscle mass (PEI following leg cycling) is there a contribution from the partial withdrawal of cardiac parasympathetic tone.