Ingestion of quinine, a bitter tastant, improves short-term (30 s) cycling performance, but it is unclear if this effect can be integrated into the last effort of a longer race. The purpose of this study was to determine if mid-trial quinine ingestion improves 3000 m cycling time trial (TT) performance. Following three familiarisation TTs, twelve well-trained male cyclists (mass 76.6 ± 9.2 kg; maximal aerobic power 390 ± 50 W; VO2 max 4.7 ± 0.6 L·min-1; mean ± SD) performed four experimental 3000 m TT on consecutive days. This double-blind cross-over design study had 4 randomized and counterbalanced conditions: 1) Quinine1 (25 ml solution, 2 mM of quinine) and 2) Quinine2, replicate of Quinine1, 3) a 25 ml sweet-tasting no carbohydrate solution (Placebo) and 4) 25 ml of water (Control) consumed at the 1850 m point of the TT. Participants completed a series of perceptual scales at the start and completion of all TTs, and power output was monitored continuously throughout all trials. Power output for the last 1000 m for all four conditions was similar: Quinine1, 360 ± 63 W (mean ± SD); Quinine2, 367 ± 63 W; Placebo, 364 ± 64 W; and Control 367 ± 58 W. There were also no differences in 3000 m TT power output between conditions. The small perceptual differences between trials at specific 150 m splits were not explained by quinine intake. Ingesting 2 mM of quinine during the last stage of a 3000 m TT did not improve cycling performance.