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We studied the effects of 24-h fasting on serum levels of guanidinoacetic acid (GAA), creatine, and creatinine in 24 non-vegetarian healthy adults. Blood was taken before and following an intervention, with levels corrected for fasting-induced changes in plasma volume. Food abstention induced a significant decrease in serum GAA by 39.5% (p < 0.001), while creatinine levels increased by 14.7% (p < 0.01); the concentration of creatine remains unchanged. The reduction in GAA during fasting may indicate an increased demand for creatine production for this energy-demanding condition and/or potential kidney dysfunction which requires further exploration.