Our objective was to determine the impact of different habituation protocols on beef cattle behavior, physiology, and temperament in response to human handling. Beef heifers were exposed to three habituation strategies: (1) tactile stimulation (brushing) in the working chute for seven consecutive days (STI; n = 18); (2) passage through the working chute for seven consecutive days (CHU; n = 19) and; (3) no habituation (CON; n = 19). Individual heifer respiratory rate (RR; n/min), internal vaginal temperature (VAGT; °C), and blood cortisol were measured. Further, behavior parameters were observed to generate a behavior score, and heifer interaction with students and their behavioral responses were recorded. Habituation with STI and CHU resulted in improved numerical behavioral scores compared to CON, and greater (p ≤ 0.05) handling latencies. Vaginal temperature was decreased in STI compared to CHU and CONT (p ≤ 0.05). Cortisol concentration did not differ among treatments, but decreased (p ≤ 0.05) from the start of the experiment to 14 days after treatment initiation. Both habituation protocols showed benefits, but heifers that received the positive tactile stimulation in the chute had the greatest behavior improvements. Furthermore, these heifers responded more calmly during student-animal interactions in class, which is beneficial for the students’ and animals’ safety.