The present study examined the effect of intensive training in combination with marked reduction in training volume on FXYD1 expression and phosphorylation at rest and during exercise. Eight well-trained cyclist replaced their regular training with speed-endurance training (10-12 x ~30-s sprints) 2-3 times per week and aerobic high-intensity training (4-5 x 3-4 min at 90-95% of peak aerobic power output) 1-2 times per week for seven weeks and reduced the training volume by 70%. Muscle biopsies were obtained before and during a repeated high-intensity exercise protocol and protein expression and phosphorylation were determined by western blotting. Expression of FXYD1 (30%), actin (40%), mTOR (12%), PLN (16%) and CaMKII γ/δ (25%) was higher (P<0.05) after compared to before the training intervention. In addition, after the intervention non-specific FXYD1 phosphorylation was higher (P<0.05) at rest and during exercise, mainly achieved by an increased FXYD1 ser68 phosphorylation, compared to before the intervention. CaMKII thr287 and eEF2 thr56 phosphorylation at rest and during exercise, overall PKCα/β thr638/641 and mTOR ser2448 phosphorylation during repeated intense exercise as well as resting PLN thr17 phosphorylation were also higher (P<0.05) after compared to before the intervention period. Thus, a period of high intensity training with reduced training volume increases expression and phosphorylation levels of FXYD1, which may affect Na(+)/K(+) pump activity and muscle K+ homeostasis during intense exercise. Furthermore, higher expression of CaMKII and PLN as well as increased phosphorylation of CaMKII thr287 may have improved intracellular Ca2+ handling.