Fatigue depresses maximal in vitro skeletal muscle Na+-K+- ATPase activity in untrained and trained individuals. J Appl Physiol 93: 1650–1659, 2002. First published July 12, 2002; 10.1152/japplphysiol.01247.2001.—This study investigated whether fatiguing dynamic exercise depresses maximal in vitro Na+-K+-ATPase activity and whether any depression is attenuated with chronic training. Eight untrained (UT), eight resistance-trained (RT), and eight endurance-trained (ET) subjects performed a quadriceps fatigue test, comprising 50 maximal isokinetic contractions (180°/s, 0.5 Hz). Muscle biopsies (vastus lateralis) were taken before and immediately after exercise and were analyzed for maximal in vitro Na+-K+-ATPase (K+-stimulated 3-O methylfluoroscein phosphatase) activity. Resting samples were analyzed for [3H]- ouabain binding site content, which was 16.6 and 18.3% higher (P < 0.05) in ET than RT and UT, respectively (UT 311 +_ 41, RT 302 +_ 52, ET 357 +_ 29 pmol/g wet wt). 3-O-methylfluoroscein phosphatase activity was depressed at fatigue by -13.8 +_ 4.1% (P < 0.05), with no differences between groups (UT -13 +_ 4, RT -9 +_ 6, ET -22 +_ 6%). During incremental exercise, ET had a lower ratio of rise in plasma K+ concentration to work than UT (P < 0.05) and tended (P = 0.09) to be lower than RT (UT 18.5 +_ 2.3, RT 16.2 +_ 2.2, ET 11.8 +_ 0.4 nmol.l -1.J-1). In conclusion, maximal in vitro Na+-K+-ATPase activity was depressed with fatigue, regardless of training state, suggesting that this may be an important determinant of fatigue.