SUMMARY Quantitative proteomics is a technique that allows for large‐scale comparison of the levels of individual proteins present in a biological sample. This technique has not previously been applied to examine the response of skeletal muscle proteins to an acute bout of exercise. In the present study, quantitative proteomics was applied to investigate whether the levels of individual skeletal muscle proteins are acutely affected by a short bout of high‐intensity exercise. Gastrocnemius muscle was sampled from fasted rats either at rest, immediately following 3 min of high‐intensity exercise or after 30 min of recovery. Muscle samples were submitted to two‐dimensional gel electrophoresis and 61 of the resulting protein spots were selected for quantitative analysis. It was found that skeletal muscle protein levels were generally not acutely affected by a short bout of high‐intensity exercise, with only four of the 61 proteins selected for analysis being significantly altered. These altered proteins were identified using liquid chromatography electrospray ionization–tandem mass spectrometry as creatine kinase, troponin T and a combination of heat shock 20 kDa protein and adenylate kinase 1. In conclusion, quantitative proteomics is sensitive enough to detect acute changes in skeletal muscle protein levels in response to exercise. We have found that the levels of most individual skeletal muscle proteins are not immediately altered in response to a short bout of high‐intensity exercise and recovery in fasted rats.