We have previously reported an acute decrease in muscle buffer capacity (βmin vitro) following high-intensity exercise. The aim of this study was to identify which muscle buffers are affected by acute exercise and the effects of exercise type and a training intervention on these changes. Whole muscle and non-protein βmin vitro were measured in male endurance athletes (VO2max = 59.8 ± 5.8 mL kg−1 min−1), and before and after training in male, team-sport athletes (VO2max = 55.6 ± 5.5 mL kg−1 min−1). Biopsies were obtained at rest and immediately after either time-to-fatigue at 120% VO2max (endurance athletes) or repeated sprints (team-sport athletes). High-intensity exercise was associated with a significant decrease in βmin vitro in endurance-trained males (146 ± 9 to 138 ± 7 mmol H+·kg d.w.−1·pH−1), and in male team-sport athletes both before (139 ± 9 to 131 ± 7 mmol H+·kg d.w.−1·pH−1) and after training (152 ± 11 to 142 ± 9 mmol H+·kg d.w.−1·pH−1). There were no acute changes in non-protein buffering capacity. There was a significant increase in βmin vitro following training, but this did not alter the post-exercise decrease in βmin vitro. In conclusion, high-intensity exercise decreased βmin vitro independent of exercise type or an interval-training intervention; this was largely explained by a decrease in protein buffering. These findings have important implications when examining training-induced changes in βmin vitro. Resting and post-exercise muscle samples cannot be used interchangeably to determine βmin vitro, and researchers must ensure that post-training measurements of βmin vitro are not influenced by an acute decrease caused by the final training bout.