Aquaporin-4 (AQP4) is expressed in skeletal muscle fast-twitch fibers. The purpose of this study was to determine whether AQP4 has a physiological role during muscle activity. Six-weeks old Wistar male rats were subjected to 5 (D5), 10 (D10) and 30 (D30) consecutive days of exhaustive exercise on a treadmill without electrical stimulation. Furthermore, CD1 AQP4-null mice were used to test whether the absence of the water channel affects voluntary and forced activity, and muscles were further used to study contractile kinetics by in vivo and ex vivo analysis. Immunoblotting and Real-Time PCR analysis performed on rat fast-twitch muscles showed a significant increase in AQP4 protein and transcripts levels in D10 and D30 rats compared to their sedentary controls, without variation in myosin heavy chain distribution and in AQP4-positive fibers percentage. In contrast, no differences were observed in D5 rats compared to the sedentaries. In particular, AQP4 up-regulation was observed in rats which ran more than 15 minutes/day, suggesting that a threshold of daily activity during a prolonged exercise (at least 10 days) must be overcome so that AQP4 over-expression occurs. Furthermore, AQP4 KO mice had significant attenuation of daily mean distances in both voluntary (-50% at D30) and forced activities (-25% at D30), even though contractile kinetics measured by ex vivo and in vivo activity were unvaried when compared to WT mice. These findings confirm that AQP4 plays a pivotal role during adaptive processes that confers the metabolic phenotype leading to improved fatigue resistance during prolonged exercise.