Chemical sedation of fish is often used during scientific research to reduce stress and risk of injury. Electric fish handling gloves (EFHG) have been proposed as a no-residue alternative to chemical immobilization. However, the impact of handling fish with EFHG on their physiology remains relatively poorly studied. Stress markers (plasma cortisol and lactate) and the impact on muscular integrity (plasma creatine kinase and histopathology of skeletal muscles) were assessed in brook trout (Salvelinus fontinalis) subjected to one of three immobilization methods (manual restraint, EFHG [10–16 mV], and buffered 60 mg/L tricaine methanesulfonate [MS-222] immersion anesthesia) for 1- and 5-min restraints. Plasma lactate levels were significantly higher 2 h postrestraint for all experimental groups (+242%; P < 0.001), but the magnitude of these increases was significantly lower in the MS-222 groups (P ≤ 0.0002). Plasma creatine kinase concentrations significantly increased 2 h postrestraint for the EFHG groups (+92%, P ≤ 0.0061), and this increase was significantly higher than in the MS-222 group for the 1-min restraint (P = 0.0031). Although there was no significant difference between the restraining methods tested at all time points for plasma cortisol, the EFHG and manual restraint groups showed a linear and statistically significant increase after the initial 5-min restraint, whereas all other groups presented an expected bell-shaped profile with a plasma peak 1 h after the initial restraint. One fish in the manual restraint group died during the 5-min restraint, and two fish from the EFHG group were euthanized at the end of this protocol because of buoyancy anomalies. All other fish from the 5-min restraint group developed cutaneous saprolegniasis 3 wk after the experiments. The evaluation of the safety of EFHG in brook trout showed similar impacts on the variables tested compared with manual restraint for 1- and 5-min immobilizations.