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Objectives The aims of this study were to detect the acetylcarnitine resonance line at 2.13 ppm in the human vastus lateralis and soleus muscles, assess T1 and T2 relaxation times, and investigate the diurnal and exercise-related changes in absolute concentration noninvasively, using proton magnetic resonance spectroscopy at 7 T. Materials and Methods All measurements were performed on a 7 T wholebody Magnetom MR system with a 28-channel knee coil. Five healthy, moderately trained volunteers participated in the assessment of the detectability, repeatability, and relaxation times of acetylcarnitine. For the evaluation of the effect of training status, another 5 healthy, normally active volunteerswere examined. In addition, normally active volunteers underwent a day-long protocol to estimate diurnal changes and response to the exercise. Results Using a long echo time of 350 milliseconds, we were able to detect the acetylcarnitine resonance line at 2.13 ppm in both muscle groups without significant lipid contamination. The T1 of acetylcarnitine in the vastus lateralis muscle was found to be 1807.2 ± 513.1 milliseconds and T2 was found to be 129.9 ± 44.9 milliseconds. Concentrations of acetylcarnitine from the vastus lateralis muscle in moderately trained volunteers were higher than concentrations from normally active volunteers. Acetylcarnitine concentrations changed during the day, tending to be higher in the morning after an overnight fast than after lunch. After 10 minutes of high-intensity exercise, the concentration significantly increased, and 15 minutes after cessation of exercise, a decrease could be observed. Conclusions Our results demonstrate an effective detection of acetylcarnitine using a long TE of 350 milliseconds at 7 T in the vastus lateralis and soleus muscles with high repeatability and reliability on a 7 T scanner. Our data emphasize the need for strict standardization, physical activity, and dietary conditions for the measurement of the acetylcarnitine.