INTRODUCTION

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RECENT STUDIES IN ISOLATED muscle preparations have demonstrated an important role for Na⁺-K⁺-ATPase in muscular fatigue, via prevention of a rundown in transmembrane Na⁺ and K⁺ gradients and thus preservation of membrane excitability (for review, see Ref. 35). Attenuation of Na⁺-K⁺-ATPase activity via inhibition with ouabain accelerates muscle fatigability and retards subsequent recovery (6), whereas, conversely, stimulation of Na⁺-K⁺-ATPase activity delays muscle fatigability and accelerates subsequent recovery in muscles paralyzed in high-K⁺ solution (6, 7, 34). Reduced Na⁺-K⁺ gradients decrease rat soleus muscle M wave area and tetanic force, whereas subsequent muscle electrical stimulation of Na⁺-K⁺-ATPase or salbutamol-induced stimulation of Na⁺-K⁺-ATPase elicited a marked recovery (39, 40). These studies highlight the importance of Na⁺-K⁺-ATPase activity in skeletal muscle function in animal models.

Muscle excitation elicits a dramatic and rapid increase above rest levels in Na⁺-K⁺-ATPase activity, measured as net Na⁺ extrusion, in isolated rat muscles (8, 33). Na⁺-K⁺-ATPase activity in isolated rat soleus muscle may increase up to 22-fold above rest after only 10 s of 120-Hz stimulation, thus approaching the maximal theoretical Na⁺-K⁺-ATPase activity (8). There are no direct measures of Na⁺-K⁺-ATPase activation in contracting human skeletal muscle. However, this is likely to also be dramatic, as shown by a rapid decline in femoral venous plasma K⁺ concentration ([K⁺]) after knee extensor exercise and by the rapid K⁺ clearance from blood after exercise (for review, see Ref. 42). Despite this increased Na⁺-K⁺-ATPase activation during muscle contractions, a direct, depressive effect of fatiguing exercise on the maximal Na⁺-K⁺-ATPase activity can be hypothesized. There is considerable structural homology of the catalytic subunits of the Ca²⁺-ATPase and Na⁺-K⁺-ATPase enzymes (20). It is now well known that fatiguing muscle contractions in humans induce an acute depression in the sarcoplasmic reticulum maximal Ca²⁺-ATPase activity or Ca²⁺-ATPase-mediated Ca²⁺ uptake rate in skeletal muscle (4, 16, 26). Hence it is conceivable that factors that adversely affect maximal Ca²⁺-ATPase activity may also impair Na⁺-K⁺-ATPase activity, and investigation into the possible effects of fatigue on Na⁺-K⁺-ATPase activity is of great interest. In human skeletal muscle, it is possible to measure the maximal in vitro Na⁺-K⁺-ATPase activity (12). However, this assay does not measure the increase in Na⁺-K⁺-ATPase activation but rather reflects the theoretical maximal Na⁺-K⁺-ATPase activity (12, 31). A recent study has indeed demonstrated depressed maximal in vitro Na⁺-K⁺-ATPase activity after repeated isometric contractions (11). However, repeated isometric contractions induce marked muscle ischemia, which might be causally linked with the impaired maximal in vitro Na⁺-K⁺-ATPase activity and the observed postcontractile depression (11). No studies thus far have investigated whether fatiguing, dynamic contractions depress maximal Na⁺-K⁺-ATPase activity in human skeletal muscle, and therefore this was the first aim of the present study.

One characteristic of training is an enhanced resistance to fatigue during activity specific to the training regimen. Factors linked with Ca²⁺-ATPase inactivation with exercise are affected by training, with modified muscle sarcoplasmic reticulum Ca²⁺ regulation during exercise by resistance and endurance training (16, 26) and increased endogenous antioxidant enzymes also evident with training (46). If the maximal Na⁺-K⁺-ATPase activity is depressed with fatigue, then chronic training could conceivably confer a protective effect on Na⁺-K⁺-ATPase activity, thereby attenuating this decline and contributing to enhanced muscular performance. Any possible protective effect of training on Na⁺-K⁺-ATPase activity may also be influenced by the total muscle Na⁺-K⁺-ATPase content, which is increased with training (9, 15, 29, 32). Thus an increased Na⁺-K⁺-ATPase content with training would offset any depressive effect of fatiguing exercise on maximal Na⁺-K⁺-ATPase activity. There have been no studies reporting both Na⁺-K⁺-ATPase content and maximal activity in trained muscle. Thus the second aim of this paper was to investigate whether chronic training protects against any possible decline in the maximal Na⁺-K⁺-ATPase activity in human muscle. No studies have examined the effects of training on maximal Na⁺-K⁺-ATPase activity per se in resting muscle in humans. The 165% increase in maximal Na⁺-K⁺-ATPase activity reported with endurance training in canine muscle (24) far exceeds the typical 14-29% increase in [³H]ouabain binding with training (31), probably because of poor and variable enzyme recovery in the isolated membrane fraction used for measurement (17). Thus the effects of chronic training on maximal Na⁺-K⁺-ATPase activity in skeletal muscle remain unknown and were also examined.

Finally, the increased muscle Na⁺-K⁺-ATPase content with training has been suggested to be important in the improved plasma K⁺ regulation during exercise, but no correlative evidence was found (15, 29). Here we explore whether muscle Na⁺-K⁺-ATPase content and maximal Na⁺-K⁺-ATPase activity are correlated to an index of plasma K⁺ regulation during exercise, the rise in plasma K⁺ concentration per unit work output (18, 29).

Three hypotheses were tested in this study: 1) that an acute bout of fatiguing dynamic exercise would depress maximal in vitro Na⁺-K⁺-ATPase activity in skeletal muscle in humans; 2) that this depression will be attenuated in chronically resistance-trained and endurance-trained athletes, and 3) that muscle Na⁺-K⁺-ATPase content and maximal Na⁺-K⁺-ATPase activity will be correlated to the rise in plasma K⁺ concentration per unit work output.

	METHODS

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Eight healthy untrained male controls (UT), eight resistance-trained subjects (RT, 7 male, 1 female), and eight endurance-trained male subjects (ET) participated after being informed of risks associated with the study and giving written, informed consent. Our laboratory has recently reported the physical characteristics, training history, anthropometry, and muscle function test results in these subjects in a paper investigating muscle Ca²⁺ regulation at rest and fatigue (26). The UT subjects were recreationally active but were not well trained and did not participate in regular sporting activities. The ET and RT athletes had been training continuously for at least 2 yr. During this period the ET athletes had performed running and/or cycling endurance training for at least 5-6 h/wk and had a peak oxygen consumption (O_2 peak) exceeding 60 ml · min¹ · kg¹. The RT subjects trained with heavy weights, typically performing three sets, six to eight repetitions, for at least 1 h and at least three sessions/wk. All were able to perform a power-lifting-style squat exercise with free weights at least 11/2 times their body mass. No significant differences existed between the three groups (means ± SD) for age (UT 26.4 ± 3.9, RT 26.8 ± 7.9, ET 26.4 ± 3.1 yr), body mass (UT 80.4 ± 6.8, RT 81.6 ± 3.3, ET 70.6 ± 9.9 kg), or height (UT 183.3 ± 5.7, RT 176.1 ± 4.7, ET 177.2 ± 7.1 cm). All protocols and procedures were approved by the Human Research Ethics Committee at Victoria University of Technology.

Overview of exercise tests. Each subject completed two tests involving invasive procedures. The first comprised an incremental cycle ergometer exercise test with arterialized-venous blood sampling conducted before, during, and after exercise. The second was a muscle fatigue test, comprising repeated maximal quadriceps contractions on an isokinetic dynamometer. A vastus lateralis muscle biopsy was taken at rest and immediately after fatiguing contractions, with arterialized venous blood sampling before, during, and after exercise. Each subject refrained from vigorous exercise, alcohol, and caffeine consumption for 24 h before each test.

Incremental exercise test. An incremental exercise test (25 W/min, 60 rpm except ET where 80 rpm) was performed on an electrically braked cycle ergometer (Lode N.V. Groningen, Netherlands) to determine O_2 peak, as detailed elsewhere (26). A catheter (20-gauge, Jelco) was inserted into a superficial dorsal hand vein of the subject before the test, and all blood samples were arterialized by heating the hand in a hot (45°C) water bath for 10 min before samples were taken (29). The catheter was kept patent by periodic infusions of heparinized isotonic saline. Arterialized venous blood was sampled at rest, in the final 10 s of each minute during graded exercise, and at 1, 2, 5, 10, 20, and 30 min in recovery, and the samples were analyzed for Hb concentration, hematocrit, as well as [K⁺] and plasma hydrogen ([H⁺]) and lactate ([Lac]) concentrations.

Muscle fatigue test. The muscle fatigue test was performed on an isokinetic dynamometer (Cybex II Lumex, Ronkoukowany), as previously detailed and justified (26). A muscle biopsy was taken at rest and immediately postexercise, and blood samples were taken at rest, mid- and immediately postexercise, and at 1, 2, 5, 10, 20, and 30 min in recovery and were analyzed as described for the incremental test. Subjects were strapped to the Cybex dynamometer chair by belts across the hips, chest, and legs to stabilize the upper body and thigh. Subjects performed 50 maximal knee extensions at a velocity of 180°/s and at a rate of 0.5 Hz (duration 100 s). Peak torque was measured and fatigue index (percentage decline in peak torque) calculated as described previously (26).

Muscle biopsy sampling and analyses. After injection of a local anesthetic into the skin and fascia (2% Xylocaine), two small incisions were made in the midportion of the vastus lateralis muscle of the right leg. The rest and fatigue biopsies were taken from separate incisions. Resting samples were analyzed for maximal in vitro Na⁺-K⁺-ATPase activity and Na⁺-K⁺-ATPase content, whereas fatigue samples were analyzed for maximal in vitro Na⁺-K⁺-ATPase activity. Muscle fiber-type composition, sarcoplasmic reticulum Ca²⁺ regulation, and metabolite contents are reported elsewhere (26).

Muscle [³H]ouabain binding site content. Approximately 20 mg of the frozen resting muscle was used to quantify the Na⁺-K⁺-ATPase content by using the [³H]ouabain binding method as previously described (21, 29, 37). Samples were cut into small pieces of 2-4 mg wet wt. In all experiments, freshly made vanadate solution was used. Samples were washed at 0°C for 20 min, with a change of medium after 10 min (2 × 10 min) in a buffer containing 10 mM Tris, 250 mM sucrose, 3 mM MgSO₄, and 1 mM vanadate, pH 7.2-7.4. This procedure was used to thaw the samples and preincubate them with vanadate and to maintain low Na⁺ and K⁺ concentrations so as not to interfere with vanadate-facilitated [³H]ouabain binding. Incubations took place in a buffer containing 2 µCi/ml [³H]ouabain and ouabain added to a final concentration of 1 µM at 37°C for 2 h, with a change of medium after 1 h. After incubations, a washout at 0°C in unlabeled buffer for 2 h with a change of medium every 30 min (4 × 30 min) was performed to reduce the [³H]ouabain in the extracellular space and enhance the precision of the method. After washout, samples were blotted on dry filter paper, weighed, and soaked overnight in minivials containing 0.5 ml of 5% trichloroacetic acid. The next day, 2.5 ml of scintillator (Opti-fluor) was added before liquid scintillation counting of the [³H]ouabain activity was performed. The amount of [³H]ouabain taken up and retained by the samples was calculated on the basis of the sample wet weight and the specific activity of the incubation medium and samples.

3-O-MFPase assay. Skeletal muscle maximal in vitro Na⁺-K⁺-ATPase activity was determined by using the K⁺-stimulated 3-O-methylfluoroscein phosphatase (3-O-MFPase) assay in human muscle homogenates, as previously described in detail (12). The 3-O-MFPase assay was chosen because it is highly sensitive, capable of determining extremely low levels of Na⁺-K⁺-ATPase activity as found in human skeletal muscle (12). This assay has two to three times higher sensitivity, therefore requiring 50-100 times less tissue, than the K⁺-stimulated p-nitro-phenyl-phosphatase (1, 36). Measurement of activity in whole muscle homogenates avoids the criticisms of very poor recovery inherent in techniques involving extensive enzymatic purification procedures (17). The assay was optimized for human skeletal muscle homogenates and specifically measures Na⁺-K⁺-ATPase activity, as evidenced by complete ouabain inhibition and K⁺ stimulation of activity (12). The interassay (5.3%) and intra-assay (8.1%) variation were acceptably low (12). Briefly, muscle samples (30-40 mg) were immediately blotted on filter paper, weighed, then homogenized (5% wt/vol) at 0°C for 2 × 20 s, 20,000 rpm (Omni 1000, Omni International) in an homogenate buffer containing 250 mM sucrose, 2 mM EDTA, and 10 mM Tris (pH 7.40). Muscle homogenates were rapidly frozen and stored in liquid nitrogen for later determination of maximal in vitro K⁺-stimulated 3-O-MFPase activity. Before analysis, homogenates were freeze-thawed four times and then diluted one-fifth in cold homogenate buffer. The assay medium in which 3-O-MFPase activity was measured contained 5 mM MgCl₂, 1.25 mM EDTA, 100 mM Tris, and an 80 nM 3-O-methyl fluorescein standard (pH 7.40). The freeze-thawed, diluted homogenate (30 µl) was incubated in 2.5 ml of assay medium at 37°C for 5 min before addition of 40 µl of 10 mM 3-O-MFP to initiate the reaction. After 60 s, 10 µl of 2.58 M KCl (final concentration 10 mM) was added to stimulate K⁺-dependent phosphatase activity, and the reaction was measured for a further 60 s. All assays were performed at 37°C, with continuous stirring, on a spectrofluorometer (Aminco Bowman AB2 SLM, Urbana, IL). Excitation wavelength was 475 nm, and emission wavelength was 515 nm, with 4-nm slit widths. The K⁺-stimulated 3-O-MFPase activity was calculated by subtracting the initial activity (comprising unspecific-ATPase activity and any spontaneous hydrolysis of 3-O-MFP) from the activity obtained after 10 mM KCl addition (12). Maximal in vitro 3-O-MFPase activity was expressed relative to muscle wet weight (nmol · min¹ · g¹ wet wt) and to identify possible effects due to fluid shifts, also relative to muscle protein content (pmol · min¹ · mg¹ protein). Protein content of the homogenate was determined spectrophotometrically by using bovine serum albumin as a standard. The relationship between [³H]ouabain binding site content and maximal in vitro 3-O-MFPase activity was examined in 22 of the 24 subjects, plus an additional six healthy untrained controls (age 40.7 ± 8.7 yr, body mass 60.8 ± 6.2 kg, height 163.9 ± 5.8 cm, means ± SD).

Blood analyses. The blood was mixed well, and air bubbles were removed from the syringe, which was capped tightly and placed on ice for subsequent duplicate analyses of plasma acid-base status and gas tensions (H⁺, PCO₂, PO₂, and [K⁺]) by use of an automated analyzer (865 Ciba Corning, Bayer). Hb concentration was determined in duplicate spectrophotometrically (Radiometer OSM2, Copenhagen, Denmark), whereas hematocrit was analyzed in triplicate after centrifugation (Hettich Zentrifugen D-7200, Tuttlingen, Germany). All analytical instruments were calibrated before and during the analyses with precision standards. An aliquot of whole blood was centrifuged at 4,000 rpm for 4 min, plasma was separated, a 200-µl aliquot of plasma was deproteinized in 600 µl of cold 3M perchloric acid, and the supernatant was later analyzed for plasma [Lac] in triplicate by use of an enzymatic spectrophotometric technique.

Calculations. The percentage decline in plasma volume from rest (PV), rise in plasma [K⁺] during exercise above rest ([K⁺]) and the ratio of [K⁺] per work done ([K⁺]/work) were calculated as previously described (29, 30), with an example of the latter as follows. If, during incremental (25 W/min) exercise, [K⁺] was 2.2 mM and the subject completed 1 min at 300 W, total work equals 117 kJ and the [K⁺]/work is 18.8 nmol · l¹ · J¹. The decline in Na⁺-K⁺-ATPase activity with fatigue is calculated as rest minus fatigue in vitro 3-O-MFPase activity. Correlations involve pooled data from all subjects in the three groups (n = 22-24, as stated).

Statistics. Data are presented as means ± SE, except population data, for which means ± SD are shown. A two-way ANOVA (sample time, group) with repeated measures (time) was used to analyze most variables. A one-way ANOVA was used when only a single variable was compared between groups (e.g., [³H]ouabain binding). Post hoc analyses used the Newman-Keuls test. Correlations between variables were determined by least-square linear regression. Significance was accepted at P < 0.05.

	RESULTS

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O_2 peak and muscle fatigue test. As a confirmation of training status, ET had a higher (P < 0.05) incremental exercise O_2 peak than the other groups (UT 44.4 ± 1.8, RT 43.8 ± 3.6, ET 67.6 ± 1.5 ml · kg¹ · min¹, means ± SD), whereas RT had a higher (P < 0.05) quadriceps maximal peak torque during isokinetic contractions from 60-300°/s (26). Peak quadriceps muscle torque declined in all groups during the 50 contractions (P < 0.05), and the fatigue index was less (P < 0.05) in ET than in UT and RT (UT 47.4 ± 14.0, RT 43.4 ± 9.4, ET 29.9 ± 12.0%, means ± SD, Ref. 26).

Muscle Na⁺-K⁺-ATPase content. The resting muscle [³H]ouabain binding site content differed between groups, being 16.6 and 18.3% higher for ET than in UT and RT, respectively (P < 0.05, Fig. 1).

View larger version (13K): [in this window] [in a new window]   Fig. 1.   Vastus lateralis muscle [3H]ouabain binding site content (Na+-K+-ATPase content) for untrained (UT), resistance-trained (RT), and endurance-trained (ET) subjects; n = 8 subjects/group (means ± SE). ET > UT; ET > RT (P < 0.05).

Maximal in vitro 3-O-MFPase activity. A significant sample time main effect was shown for maximal in vitro 3-O-MFPase activity expressed per gram wet weight, with a decline of 13.8 ± 4.1% at fatigue (Fig. 2A, P < 0.05). No significant group main effects or time-by-group interactions were seen, although resting 3-O-MFPase activity in ET tended to be higher (20.3%) than in UT. The decline in maximal in vitro 3-O-MFPase activity at fatigue did not differ significantly between the groups (absolute: UT 27 ± 8, RT 24 ± 13, ET 60 ± 17 nmol · min¹ · g wet wt¹; relative: UT 13 ± 4, RT 9 ± 6, ET 22 ± 6%).

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Maximal in vitro K+-stimulated 3-O-methylfluoroscein phosphatase (3-O-MFPase) activity (Na+-K+-ATPase activity) in skeletal muscle obtained at rest and fatigue for UT, RT, and ET subjects. A: activity expressed per gram weight wet. B: activity expressed per milligram protein. Data are means ± SE; n = 7 subjects for UT and ET and n = 8 subjects for RT. * Main effect of exercise, fatigue < rest (P < 0.05).

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Plasma electrolytes and plasma volume changes during the muscle fatigue test. A significant time main effect was found for arterialized-venous plasma [K⁺] (P < 0.05), which increased midexercise (P < 0.05), peaked at fatigue, and returned to rest values by 2 min recovery (Fig. 3A). No significant group main effect or time-by-group interactions were found for plasma [K⁺]. No between-group differences were found during the fatigue test for peak plasma [K⁺] (UT 4.81 ± 0.17, RT 4.57 ± 0.17, ET 4.60 ± 0.09 mmol/l), [K⁺] (UT 0.92 ± 0.13, RT 0.60 ± 0.12, ET 0.85 ± 0.09 mmol/l), or [K⁺]/work (UT 85.7 ± 13.0, RT 59.7 ± 11.6, ET 77.6 ± 8.5 nmol · l¹ · J¹).

View larger version (19K): [in this window] [in a new window]   Fig. 3.   Arterialized-venous plasma K+ concentration ([K+]) for UT (), RT (), and ET () subjects during muscle fatigue test (A) and during recovery incremental test (B). In A, plasma K+ concentration ([K+]) is shown at rest (R), midexercise (M), and end exercise (E). In B, plasma [K+] is shown at rest, each minute during exercise, and during 30 min of recovery. In B, horizontal error bars indicate mean ± SE peak work rate. The peak incremental exercise plasma [K+] is replotted as the zero-recovery time point. * Main effect of exercise time (A) and/or work rate (B) [K+] > rest (P < 0.05). Data are means ± SE; n = 8 for UT and ET, n = 7 for RT.

View this table: [in this window] [in a new window]   Table 1.   PV, plasma [lactate], and [H+] during the quadriceps muscle fatigue test in UT, RT, and ET subjects

Plasma electrolytes and plasma volume changes during the incremental exercise test. A significant time main effect was found for arterialized-venous plasma [K⁺] (P < 0.05), which increased above rest from 75 W until the peak incremental exercise work rate and had returned to rest by 5 min recovery (Fig. 3B). No significant group main effect or time-by-group interactions were found for plasma [K⁺]. No between-group differences were found for peak plasma [K⁺] (UT 6.14 ± 0.17, RT 6.07 ± 0.11, ET 6.43 ± 0.25 mmol/l) or [K⁺] (UT 2.20 ± 0.16, RT 2.11 ± 0.15, ET 2.42 ± 0.19 mmol/l), but the [K⁺]/work was 36% lower in ET (11.8 ± 0.4 nmol · l¹ · J¹) compared with UT (18.5 ± 2.3 nmol · l¹ · J¹, P < 0.05) and also tended to be lower than in RT (16.2 ± 2.2 nmol · l¹ · J¹, P = 0.09).

View this table: [in this window] [in a new window]   Table 2.   Plasma volume changes from rest (PV), plasma [lactate] and [H+] during an incremental cycle ergometer exercise test in UT, RT, and ET subjects

Functional correlates of muscle 3-O-MFPase activity and Na⁺-K⁺-ATPase content. In resting muscle samples, a significant correlation was found between [³H]ouabain binding site content and the maximal in vitro 3-O-MFPase activity (Fig. 4A, r = 0.61, n = 28, P < 0.05). The [³H]ouabain binding site content was inversely correlated with the fatigue index (Fig. 4B, r = 0.42, n = 24, P < 0.05) and correlated with O_2 peak (Fig. 5A, r = 0.64, n = 23, P < 0.05). The maximal in vitro 3-O-MFPase activity was also correlated with O_2 peak (Fig. 5B, r = 0.46, n = 22, P < 0.05) but not with the fatigue index (r = 0.24, NS).

View larger version (15K): [in this window] [in a new window]   Fig. 4.   Relationship between [3H]ouabain binding site content (Na+-K+-ATPase content; pmol/g wet wt) and 3-O-MFPase activity (Na+-K+-ATPase activity; nmol · min1 · g1 wet wt, n = 28) (A) and fatigue index during the muscle fatigue test (%, n = 24) (B) in UT (), RT (), ET (), and age-matched controls ().

View larger version (13K): [in this window] [in a new window]   Fig. 5.   Relationship between peak O2 consumption (O2 peak; l/min) and 3-O-MFPase activity (Na+-K+-ATPase activity, nmol min1 · g1 wet wt, n = 22) (A) and Na+-K+-ATPase content (pmol/g wet wt, n = 23) (B) for pooled UT (), RT (), and ET () subjects.

View larger version (14K): [in this window] [in a new window]   Fig. 6.   Relationships between the incremental exercise ([K+]/work; nmol · l1 · J1) and maximal in vitro 3-O-MFPase activity (Na+-K+-ATPase activity; nmol · min1 · g1 wet wt, n = 22) (A) and [3H]ouabain binding site content (Na+-K+-ATPase content; pmol/g wet wt, n = 24) (B) for UT (), RT (), and ET () subjects.

	DISCUSSION

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Fatigue depresses muscle maximal Na+-K⁺-ATPase activity. The most important and novel finding of this study was that an acute bout of fatiguing dynamic exercise depressed the skeletal muscle maximal in vitro 3-O-MFPase activity, which is a measure of Na⁺-K⁺-ATPase activity. The similar decline at fatigue in 3-O-MFPase activity expressed relative to muscle wet weight or protein content argues strongly against the possibility of an artifactual effect due to a contraction-induced vascular fluid shift into muscle. Although we did not measure [³H]ouabain binding site content in the fatigued sample, it seems improbable that a loss of Na⁺-K⁺-ATPase pump units could occur in this time frame. Rather, an inactivation of these pump units is the more likely explanation. This is the first time such a depression has been demonstrated with dynamic exercise and implicates Na⁺-K⁺-ATPase as an additional potential site for muscle fatigue during intense muscle contractions.

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Functional implications for Na⁺-K⁺-ATPase: muscle performance and plasma [K⁺]. We demonstrate an important functional role of Na⁺-K⁺-ATPase for muscle contractile performance in humans via relationships between the maximal in vitro 3-O-MFPase activity, [³H]ouabain binding site content, and two indexes of dynamic muscular performance: fatigability during repeated quadriceps contractions and the peak incremental exercise O₂ uptake. Both the maximal in vitro 3-O-MFPase activity and the [³H]ouabain binding site content were significantly correlated with O_2 peak. A novel finding in human muscle was the significant inverse relationship between the [³H]ouabain binding site content and fatigability. This is consistent with studies in rat muscle in which Na⁺-K⁺-ATPase activation correlated with contractile performance (7, 33), although other studies in humans have failed to find a relationship between Na⁺-K⁺-ATPase content and muscle endurance during fatiguing isometric contractions (23) or repeated sprints (29). Others found depressions in each of muscle isometric force, M-wave area, and maximal in vitro 3-O-MFPase activity after isometric contractions, although these were not reported as being directly linked (11). Surprisingly, we did not find a positive relationship between either the maximal in vitro 3-O-MFPase activity or the fatigue-induced decline in 3-O-MFPase activity with the fatigue index. This may reflect variability in the 3-O-MFPase activity measures but is also consistent with multiple additional factors contributing to muscle fatigue, including impaired sarcoplasmic reticulum Ca²⁺ release and uptake (4, 26), K⁺ loss (42), metabolic perturbations such as increased intracellular P_i (10), and fatigue of the central nervous system (13).