Background Intravenous high-dose glucagon is a recommended antidote against beta-blocker poisonings, but clinical effects are unclear. We therefore investigated hemodynamic effects and safety of high-dose glucagon with and without concomitant beta-blockade. Methods and Results In a randomized crossover study, 10 healthy men received combinations of esmolol (1.25 mg/kg bolus+0.75 mg/kg/min infusion), glucagon (50 µg/kg), and identical volumes of saline placebo on 5 separate days in random order (saline+saline; esmolol+saline; esmolol+glucagon bolus; saline+glucagon infusion; saline+glucagon bolus). On individual days, esmolol/saline was infused from -15 to 30 minutes. Glucagon/saline was administered from 0 minutes as a 2-minute intravenous bolus or as a 30-minute infusion (same total glucagon dose). End points were hemodynamic and adverse effects of glucagon compared with saline. Compared with saline, glucagon bolus increased mean heart rate by 13.0 beats per minute (95% CI, 8.0-18.0; P<0.001), systolic blood pressure by 15.6 mm Hg (95% CI, 8.0-23.2; P=0.002), diastolic blood pressure by 9.4 mm Hg (95% CI, 6.3-12.6; P<0.001), and cardiac output by 18.0 % (95% CI, 9.7-26.9; P=0.003) at the 5-minute time point on days without beta-blockade. Similar effects of glucagon bolus occurred on days with beta-blockade and between 15 and 30 minutes during infusion. Hemodynamic effects of glucagon thus reflected pharmacologic glucagon plasma concentrations. Glucagon-induced nausea occurred in 80% of participants despite ondansetron pretreatment. Conclusions High-dose glucagon boluses had significant hemodynamic effects regardless of beta-blockade. A glucagon infusion had comparable and apparently longer-lasting effects compared with bolus, indicating that infusion may be preferable to bolus injections. Registration Information URL: https://www.clinicaltrials.gov; Unique identifier: NCT03533179.