The goal of this study was to assess the effects of repeated running bouts with end-expiratory breath holding (EEBH) up to the breaking point on muscle oxygenation. Eight male runners participated in three randomised sessions each including two exercises on a motorised treadmill. The first exercise consisted in performing 10–12 running bouts with EEBH of maximum duration either (separate sessions) at 60% (active recovery), 80% (passive recovery) or 100% (passive recovery) of the maximal aerobic velocity (MAV). Each repetition started at the onset of EEBH and ended at its release. In the second exercise of the session, subjects replicated the same procedure but with normal breathing (NB). Arterial oxygen saturation (SpO2), heart rate (HR) and the change in vastus lateralis muscle deoxy-haemoglobin/myoglobin ($Δ$[HHb/Mb]) and total haemoglobin/myoglobin ($Δ$[THb/Mb]) were continuously monitored throughout exercises. On average, the EEBHs were maintained for 10.1 ± 1.1 s, 13.2 ± 1.8 s and 12.2 ± 1.7 s during exercise at 60%, 80% and 100% of MAV, respectively. In the three exercise intensities, SpO2 (mean nadir values: 76.3 ± 2.5 vs 94.5 ± 2.5%) and HR were lower with EEBH than with NB at the end of the repetitions; whereas, the mean $Δ$[HHb/Mb] (12.6 ± 5.2 vs 7.7 ± 4.4 µm) and $Δ$[THb/Mb] (− 0.6 ± 2.3 vs 3.8 ± 2.6 µm) were, respectively, higher and lower with EEBH (p < 0.05). This study showed that performing repeated bouts of running exercises with EEBH up to the breaking point induced a large and early drop in muscle oxygenation compared with the same exercise with NB. This phenomenon was probably the consequence of the strong arterial oxygen desaturation induced by the maximal EEBHs.