Purpose This study evaluated the impact of limited and strenuous physical exercise on [18F]methylcholine uptake in muscle. Methods Ten consecutive patients participated, three of whom had strict bed rest, three were allowed to walk around and four performed strenuous single arm exercise by lifting a 7.5-kg weight. [18F]Methylcholine uptake was measured in the biceps and gluteus muscles on both sides. Results Strenuous exercise resulted in a 202% increase in [18F]methylcholine uptake in the activated biceps muscle as well as a 112% increase in muscle groups used to retain body position. This resulted in asymmetrical images that were visually less easy to interpret. In walking patients there was a more limited increase in biceps (45%) and gluteus (74%) muscle uptake, without visually recognizable differences. Conclusion Strenuous exercise may result in a considerable increase in [18F]methylcholine uptake in muscle and should be avoided prior to imaging. Strict bed rest does not seem to be required. Tracer injection while resting on the scanner remains a safe approach.