Purpose: This study aimed to determine the neuro-mechanical and metabolic adjustments in the lower limbs induced by the running anaerobic sprint test (the so-called RAST). Methods: Eight professional football players performed 6 × 35 m sprints interspersed with 10 s of active recovery on artificial turf with their football shoes. Sprinting mechanics (plantar pressure insoles), root mean square activity of the vastus lateralis (VL), rectus femoris (RF), and biceps femoris (BF) muscles (surface electromyography, EMG) and VL muscle oxygenation (near-infrared spectroscopy) were monitored continuously. Results: Sprint time, contact time and total stride duration increased from the first to the last repetition (+17.4, +20.0 and +16.6 %; all P < 0.05), while flight time and stride length remained constant. Stride frequency (−13.9 %; P < 0.001) and vertical stiffness decreased (−27.2 %; P < 0.001) across trials. Root mean square EMG activities of RF and BF (−18.7 and −18.1 %; P < 0.01 and 0.001, respectively), but not VL (−1.2 %; P > 0.05), decreased over sprint repetitions and were correlated with the increase in running time (r = −0.82 and −0.90; both P < 0.05). Together with a better maintenance of RF and BF muscles activation levels over sprint repetitions, players with a better repeated-sprint performance (lower cumulated times) also displayed faster muscle de- (during sprints) and re-oxygenation (during recovery) rates (r = −0.74 and −0.84; P < 0.05 and 0.01, respectively). Conclusion: The repeated anaerobic sprint test leads to substantial alterations in stride mechanics and leg-spring behaviour. Our results also strengthen the link between repeated-sprint ability and the change in neuromuscular activation as well as in muscle de- and re-oxygenation rates.