The Wim Hof breathing method (WHBM) combines periods of hyperventilation (HV) followed by voluntary breath-holds (BH) at low lung volume. It has been increasingly adopted by coaches and their athletes to improve performance, but there was no published research on its effects. We determined the feasibility of implementing a single WHBM session before repeated sprinting performance and evaluated any acute ergogenic effects. Fifteen amateur runners performed a single WHBM session prior to a Repeated Ability Sprint Test (RAST) in comparison to voluntary HV or spontaneous breathing (SB) (control) in a randomized cross-over design. Gas exchange, heart rate, and finger pulse oxygen saturation (SpO₂) were monitored. Despite large physiological effects in the SpO₂ and expired carbon dioxide (VCO₂) levels of both HV and WHBM, no significant positive or negative condition effects were found on RAST peak power, average power, or fatigue index. Finger SpO₂ dropped to 60 ± 12% at the end of the BHs. Upon the last HV in the WHBM and HV conditions, end-tidal CO₂ partial pressure (PETCO₂) values were 19 ± 3 and 17 ± 3 mmHg, indicative of respiratory alkalosis with estimated arterial pH increases of +0.171 and of +0.181, respectively. Upon completion of RAST, 8 min cumulated expired carbon dioxide volumes in the WHBM and HV were greater than in SB, suggesting lingering carbon dioxide stores depletion. These findings indicate that despite large physiological effects, a single WHBM session does not improve anaerobic performance in repeated sprinting exercise.