An automated respirometer system was used to measure VO2, protein catabolism as ammonia quotient and the energy budget to evaluate whether the crude protein content of a standard protein (SP) diet (42·5 %) or a high-protein (HP) diet (49·5 %) influences metabolism in rainbow trout under challenging intermittent, low dissolved oxygen concentrations. In total, three temperature phases (12, 16, 20°C) were tested sequentially, each of which were split into two oxygen periods with 5 d of unmanipulated oxygen levels (50–70 %), followed by a 5d manipulated oxygen period (16.00–08.00 hours) with low oxygen (40–50 %) levels. For both diets, catabolic protein usage was lowest at 16°C and was not altered under challenging oxygen conditions. Low night-time oxygen elevated mean daily VO2 by 3–14 % compared with the unmanipulated oxygen period for both diets at all temperatures. The relative change in VO2 and retained energy during the intermittent low oxygen period was smaller for the HP diet compared with the SP diet. However, in absolute terms, the SP diet was superior to the HP diet as the former demonstrated 30–40 % lower protein fuel use rates, higher retained energy (1–4 % digestible energy) and slightly lowered VO2 (0–8 %) over the range of conditions tested. The decrease in retained energy under low oxygen conditions suggests that there is scope to improve the performance of SP diets under challenging conditions; however, this study suggests that simply increasing the dietary protein content is not a remedy, and other strategies need to be explored.