Prior research did not provide data on acute adaptations to changes in saddle height as commonly performed in bicycle fitting. This study assessed the acute responses from recreational cyclists to changes in saddle height using dynamic bicycle fitting. Ten recreational cyclists completed five trials using three saddle heights (Preferred, High and Low), one Sham-height trial and a final repeated trial for assessment of intra-session reliability. High and Low saddle heights were determined based on dynamically measured knee flexion angles (±10° from their Preferred height). Trials were performed for 30-s at 90 ± 2 rpm and 2.5 W/kg (181 ± 32 W). Perceived comfort and discomfort and rating of perceived exertion were assessed at the end of each trial. Significant differences were determined when p < 0.05. Increased comfort was observed at the preferred saddle height compared to a lower saddle height ( p = 0.03) but no differences were observed between Preferred and High ( p = 0.69) or High vs. Low ( p = 0.06). The Sham height was also not different from its equivalent saddle height ( p = 0.77) and rating of perceived exertion did not differ between saddle heights ( p = 0.33). Recreational cyclists were generally sensitive to changes in saddle height given they reported different levels of comfort between their preferred and a lower saddle height. Similar levels of comfort were observed when a Sham height was introduced, showing that recreational cyclists identified that no change in saddle height was conducted. However, these cyclists could not report differences in comfort when cycling at a High compared to a Low saddle height.