Three experiments were performed to examine the effects of high ambient temperature and feed restrictions on urinary 3-methylhistidine (3MH) excretion and plasma 3MH concentrations as a marker of myofibrillar proteolysis in lactating Holstein cows. In a crossover design, four cows were maintained during two 14-day treatment periods with ad libitum feed intake under constant moderate (18 degrees C) or high (28 degrees C) ambient temperature (Experiment 1), with ad libitum or 70% ad libitum feed intake under constant moderate temperature (Experiment 2), and with 70% ad libitum feed intake under constant moderate or high ambient temperature (Experiment 3). The total digestible nutrients intake was decreased (P < 0.01) under high ambient temperature (Experiment 1) or feed restriction (Experiment 2). Across experiments, urinary 3MH excretion during days 10-14 of each treatment period was not different among treatments. However, the plasma 3MH concentrations at day 14 of treatment were increased significantly under high ambient temperature with subsequent reduced feeding (P < 0.01) or moderate temperature with feed restriction (P < 0.05), and were increased slightly (P = 0. 11) under high ambient temperature alone. These results show that the plasma marker of myofibrillar proteolysis in lactating dairy cows was increased at day 14 of treatments of high ambient temperature with subsequent reduced feeding, moderate temperature with feed restriction, or high ambient temperature alone.