A public debate has recently arisen, largely surrounding the issue of pain, over whether freeze or hot-iron branding should be the preferred method of permanently identifying cattle. This study addressed that question by quantifying the following accepted measures of distress and pain over a 25-min sampling period: elevated heart rate, concentrations of Cortisol, epinephrine, and norepinephrine, and escape-avoidance reactions and vocalizations. Twenty-four dairy cows (15 Holsteins and 9 Jerseys) were assigned to one of three treatments: freeze-branded (F), hot-iron-branded (H), or sham-branded (S), in which a room-temperature brander was applied. Plasma epinephrine and norepinephrine concentrations showed no discernible trends. Plasma Cortisol concentrations were elevated in the F and H cows from 5.5 min to 25.5 min postbranding (P = .04). Heart rate, analyzed as a proportion of the prebranding mean, showed that H cows had a greater, more acute, response than did F cows (P = .04), which exhibited a more prolonged response (P = .07). No cows vocalized during branding; however, H cows had a greater escape-avoidance reaction toward branding than did the F and S cows. Both methods of branding produced elevated heart rates and Cortisol concentrations indicative of pain sensations. Because the cows exhibited a greater escape-avoidance reaction and heart rate proportions to hot-iron branding, freeze banding would be preferable to hot-iron branding when feasible.