Due to the current lack of clarity, we examined whether 5 days of dietary creatine (Cr) supplementation per se can influence the glycogen content of human skeletal muscle. Six healthy male volunteers participated in the study, reporting to the laboratory on 4 occasions to exercise to the point of volitional exhaustion, each after 3 days of a controlled normal habitual dietary intake. After a familiarisation visit, participants cycled to exhaustion in the absence of any supplementation (N), then two weeks later again cycled to exhaustion after 5 days of supplementation with simple sugars (CHO). Finally, after a further 2 weeks they again cycled to exhaustion after 5 days of Cr supplementation. Muscle samples were taken at rest before exercise, and at the time-point of exhaustion in visit 1, and at subsequent visit time of exhaustion. There was a treatment effect on muscle total Cr content in Cr compared with N and CHO supplementation (P < 0.01). Resting muscle glycogen content was elevated above N following CHO (P < 0.05), but not after Cr. At exhaustion following N, glycogen content was no different from CHO and Cr measured at the same time point during exercise. Creatine supplementation under conditions of controlled habitual dietary intake had no effect on muscle glycogen content at rest or after exhaustive exercise. We suggest that any Cr associated increases in muscle glycogen storage are the result of an interaction between creatine supplementation and other mediators of muscle glycogen storage.