Objective To examine the effect of pre-cooling (PreC) on cycling time-trial (CTT) performance in heat, before and after heat acclimation (HA). Design Randomised crossover. Methods Ten trained/highly trained male cyclists and/or triathletes completed two 20-km CTT before (PreHA) HA training sessions (10 × 60 min intermittent-heat exposure protocol in 36°C, 50-80% relative humidity), and after (PostHA). No cooling (CON) or crushed-ice was ingested (i.e., PreC) 30min prior to the CTTs. The first and final HA training sessions were matched and acted as heat stress tests for comparison. Results No meaningful direct relations were observed for 20-km CTT completion time between PostHA+PreC (2663 ± 307s) and PostHA-CON (2671 ± 370s; b = 37.81[-109.98,170.56]). Split times were faster in the first 12.5km of the CTT in PostHA+PreC but slower across the rest of the CTT compared to PostHA-CON (b = -1.224[-2.196,-0.157]). Core temperature was lower in PostHA-CON compared to PostHA+PreC (b = -0.02[-0.04,-0.01]). No difference were observed for mean skin temperature (b = -0.16[-0.27,-0.05]) and thermal sensation (b = -0.047[-0.091,-0.003]) during the CTT. Conclusions. Insufficient evidence exists to support a meaningful performance improvement in 20-km CTT in hot-humid conditions when PreC was applied to individuals who completed a HA regime. This may be attributed to the limited effect of PreC on thermal perception, potentially leading to decreased exercise intensity in the latter stages of the CTT as a strategy to mitigate heat gain. Additionally, sub-optimal pacing strategies resulting from PreC on individuals may explain the lack of additional benefit to performance. Abbreviations PreC (Pre-cooling), CON (Control), HA (Heat acclimation), PreHA (Pre-heat acclimation), PostHA (Post-heat acclimation), USG (Urine specific gravity), CTT (Cycling time-trial), Tc (Core temperature), Tsk (Skin temperature), Tb (Body temperature), Tg (Core-to-skin temperature gradient), Thc (Thermal comfort), Ths (Thermal sensation), RPE (Rating of perceived exertion), HeatAcc (Heat acclimated/acclimatised),
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