Methylphenidate (MPD) is one of the most prescribed pharmacological agents, which is also used for cognitive enhancement and recreational purposes. The objective of this study was to investigate the repetitive dose-response effects of MPD on circadian rhythm of locomotor activity pattern of female WKY rats. The hypothesis is that a change in the circadian activity pattern indicates a long-lasting effect of the drug. Four animal groups (saline control, 0.6, 2.5, and 10.0 mg/kg MPD dose groups) were housed in a sound-controlled room at 12:12 light/dark cycle. All received saline injections on experimental day 1 (ED 1). On EDs 2-7, the control group received saline injection; the other groups received 0.6, 2.5, or 10.0 mg/kg MPD, respectively. On ED 8-10, injections were withheld. On ED 11, each group received the same dose as EDs 2-7. Hourly histograms and cosine statistical analyses calculating the acrophase (ϕ), amplitude (A), and MESOR (M) were applied to assess the 24-h circadian activity pattern. The 0.6 and 2.5 mg/kg MPD groups exhibited significant (p < 0.05) change in their circadian activity pattern on ED 11. The 10.0 mg/kg MPD group exhibited tolerance on ED 11 and also a significant change in activity pattern on ED 8 compared to ED 1, consistent with withdrawal behavior (p < 0.007). In conclusion, chronic MPD administration alters circadian locomotor activity of adult female WKY rats and confirms that chronic MPD use elicits long-lasting effects.