Haematological parameters decrease following one unit of whole blood donation which results in a detrimental effect on cardiorespiratory fitness and maximal exercise capacity. However, it remains unclear to what extent blood donation will impact exercise performance across varying levels of cardiorespiratory fitness. The aim of this study is to compare the effects of a standard blood donation on maximal exercise performance performed 24 h post-blood donation in individuals with varying levels of cardiorespiratory fitness. Methods: Forty-two males (mean age 22 ± 2.1 years) were recruited and segregated into Low, Moderate and Superior fitness groups. Subjects performed a multi-stage shuttle run test on the day prior to a standard blood donation procedure, and subsequently 24 h post-blood donation. Blood samples were taken on both test days and analysed for haematological parameters. Results: Exercise performance, represented by predicted VO2max were 0.6%, 1.0% and 4.1% lower in the Low, Moderate and Superior fitness groups respectively. However, the magnitude of reduction was only statistically significant in the Superior fitness group (p = 0.017). Compared to baseline, all fitness groups demonstrated significant reductions in haematocrit (Low: -8.4%, Moderate: -9.1%, Superior: -7.2%) and haemoglobin (Low: -7.6%, Moderate: -7.8%, Superior: -5.5%) levels at 24 h post-blood donation. Spearman correlation analysis revealed that changes in haemoglobin concentrations were not associated with changes in exercise performance in all groups. Conclusion: Exercise performance was only significantly reduced in the Superior fitness group. Well-trained individuals should be refrained from blood donation just prior to competitions.