Objective: To determine the effect of transportation during periods of high ambient temperature on physiologic and behavioral indices of beef heifers. Animals: 20 heifers (mean body weight, 217.8 kg). Procedures: Ten heifers were transported 518 km when the maximum ambient temperature was ≥ 32.2°C while the other 10 heifers served as untransported controls. Blood samples were collected from transported heifers at predetermined intervals during the transportation period. For all heifers, body weights, nasal and rectal temperatures, and behavioral indices were measured at predetermined intervals for 3 days after transportation. A week later, the entire process was repeated such that each group was transported twice and served as the control twice. Results: Transported heifers spent more time near the hay feeder on the day of transportation, had lower nasal and rectal temperatures for 24 hours after transportation, and spent more time lying down for 2 days after transportation, compared with those indices for control heifers. Eight hours after transportation, the weight of transported heifers decreased 6%, whereas that of control heifers increased 0.6%. At 48 hours after initiation of transportation, weight, rectal temperature, and time spent at various pen locations did not differ between transported and control heifers. Cortisol concentrations were higher 4 hours after initiation of transportation, compared with those determined just prior to transportation. Conclusions and Clinical Relevance: Results indicated transportation during periods of high ambient temperatures caused transient changes in physiologic and behavioral indices of beef heifers.