A rise in extracellular potassium concentration in human skeletal muscle may play an important role in development of fatigue during intense exercise. The aim of the present study was to examine the effect of intense intermittent training on muscle interstitial potassium kinetics and its relationship to the density of Na⁺,K⁺‐ATPase subunits and K_ATP channels, as well as exercise performance, in human skeletal muscle. Six male subjects performed intense one‐legged knee‐extensor training for 7 weeks. On separate days the trained leg (TL) and the control leg (CL) performed a 30 min exercise period of 30 W and an incremental test to exhaustion. At frequent intervals during the exercise periods interstitial potassium ([K⁺]_I) was determined by microdialysis, femoral arterial and venous blood samples were drawn and thigh blood flow was measured. Time to fatigue for TL was 28% longer (P < 0.05) than for CL (10.6 ± 0.7 (mean ±s.e.m.) versus 8.2 ± 0.7 min). The amounts of Na⁺,K⁺‐ATPase α₁ and α₂ subunits were, respectively, 29.0 ± 8.4 and 15.1 ± 2.7% higher (P < 0.05) in TL than in CL, while the amounts of β₁ subunits and ATP‐dependent K⁺ (K_ATP) channels were the same. In CL [K⁺]_I increased more rapidly and was higher (P < 0.05) throughout the 30 W exercise bout, as well at 60 and 70 W, compared to TL, whereas [K⁺]_I was similar at the point of fatigue (9.9 ± 0.7 and 9.1 ± 0.5 mmol l⁻¹, respectively). During the 30 W exercise bouts and at 70 W during the incremental exercise femoral venous potassium concentration ([K⁺]_v) was higher (P < 0.05) in CL than in TL, but identical at exhaustion (6.2 ± 0.2 mmol l⁻¹). Release of potassium to the blood was not different in the two legs. The present data demonstrated that intense intermittent training reduce accumulation of potassium in human skeletal muscle interstitium during exercise, probably through a larger re‐uptake of potassium due to greater activity of the muscle Na⁺,K⁺‐ATPase pumps. The lower accumulation of potassium in muscle interstitium in the trained leg was associated with delayed fatigue during intense exercise, supporting the hypothesis that interstitial potassium accumulation is involved in the development of fatigue.