In animals, the blockade of acid-sensing ion channels (ASICs), cation pore-forming membrane proteins located in the free nerve endings of group IV afferent fibres, attenuates increases in arterial pressure (AP) and sympathetic nerve activity (SNA) during muscle contraction. Therefore, ASICs play a role in mediating the metabolic component (skeletal muscle metaboreflex) of the exercise pressor reflex in animal models. Here we tested the hypothesis that ASICs also play a role in evoking the skeletal muscle metaboreflex in humans, quantifying beat-by-beat mean AP (MAP, finger photoplethysmography) and muscle SNA (MSNA, microneurography) in 11 men at rest, during static handgrip exercise (SHE, 35% of the maximal voluntary contraction) and post-exercise muscle ischemia (PEMI) before (B) and after (A) local venous infusion of either saline or amiloride (AM), an ASICs antagonist, via the Bier blocktechnique. MAP (BAM +30 ± 6 vs. AAM +25 ± 7 mmHg, p = 0.001) and MSNA (BAM +14 ± 9 vs. AAM +10 ± 6 bursts/min, p = 0.004) responses to SHE were attenuated under ASICs blockade. Amiloride also attenuated the PEMI-induced increases in MAP (BAM +25 ± 6 vs. AAM +16 ± 6 mmHg, p = 0.0001) and MSNA (BAM +16 ± 9 vs. AAM +8 ± 8 bursts/min, p = 0.0001). MAP and MSNA responses to SHE and PEMI were similar before and after saline infusion. We conclude that ASICs play a role in evoking pressor and sympathetic responses to SHE and the isolated activation of the skeletal muscle metaboreflex in humans.