The aim of the present study is to investigate the effect of acetic acid feeding on the circadian changes in glycogen concentration in liver and skeletal muscle. Rats were provided meal once daily (09.00–13.00 hours) for 10d. On the 11th day, they were either killed immediately or given 9g diet containing either 0 (control) or 0·7g/kg-diet acetic acid beginning at 09.00 hours for 4h, as in the previous regimen. Rats in the fed group were killed at 4, 8 or 24h after the start of feeding. At 4h after the start of feeding, the acetic acid group had significantly greater liver and gastrocnemius muscle glycogen concentrations (P<0·05). Also, at this same point, liver xylulose-5-phosphate, a key stimulator of glycolysis, the ratio of fructose-1,6-bisphosphate to fructose-6-phosphate in skeletal muscle, which reflects phosphofructokinase-1 activity, and liver malonyl-CoA, an allosteric inhibitor of carnitine palmitoyl-transferase, were significantly lower in the acetic acid group than in the control group (P<0·05). In addition, the acetic acid group had a significantly lower serum lactate concentration and lower ratio of insulin to glucagon than the control group at the same point (P<0·05). We conclude that a diet containing acetic acid may enhance glycogen repletion but not induce supercompensation, a large increase in the glycogen level that is beneficial in improving performance, in liver and skeletal muscle by transitory inhibition of glycolysis. Further, we indicate the possibility of a transient enhancement of fatty acid oxidation in liver by acetic acid feeding.