Caffeine increases aerobic performance, but research findings are mixed regarding anaerobic performance (AnP). Thus, the purpose of this study was to investigate the effects of acute caffeine ingestion on high-intensity cycling anaerobic exercise in 14 healthy and physically active non-habitual caffeine users (mean age: 20.93 ± 1.73 yrs). After the subjects were familarized with the Wingate Anaerobic test (WAnT) intensity (on a separate date), placebo or caffeine treatments were performed. Both treatments were separated by 1 wk in a randomized, double-blind manner. One hr before the WAnT, the subjects ingested a drink mixture of water, lemonade, and Sprite as a placebo (PLA) or containing caffeine (CAF, 5 mg·kg-1); both drinks were equal in calories. The WAnT was performed for 30 sec with the resistance set at 7.5% of the subject's body mass. Peak power, mean power, and fatigue index were determined. A paired sample t-test was used to compare the AnP variables between treatments. Heart rate (HR), blood pressure (BP), blood lactate (LA), and glucose (GLU) levels were analyzed with a two-way repeated-measures ANOVA. The results showed no significant effect of caffeine on AnP (P > 0.05). Increases in HR, BP, and blood LA levels were independent of treatments (P > 0.05). However, blood GLU levels significantly differed between the treatments (P < 0.05). The findings indicate that a single dose of CAF was insufficient to elicit an ergogenic effect on AnP in unaccustomed and non-habitual caffeine users.