Turning down lipid oxidation during heavy exercise--what is the mechanism?

A high potential for lipid oxidation is a sign of metabolic fitness and is important not only for exercise performance but also for health promotion. Despite considerable progress during recent years, our understanding of how lipid oxidation is controlled remains unclear. The rate of lipid oxidation reaches a peak at 50-60% of V(O2 max) after which the contribution of lipids decreases both in relative and absolute terms. In the high-intensity domain (>60% V(O2 max)), there is a pronounced decrease in energy state, which will stimulate the glycolytic rate in excess of the substrate requirements of mitochondrial oxidative processes. Accumulation of glycolytic products will impair lipid oxidation through an interaction with the carnitine-mediated transfer of FA into mitochondria. Another potential site of control is Acyl-CoA synthetase (ACS), which is the initial step in FA catabolism. The activity of ACS may be under control of CoASH and energy state. There is evidence that additional control points exist beyond mitochondrial influx of fatty acids. The electron transport chain (ETC) with associated feed-back control by redox state is one suggested candidate. In this review it is suggested that the control of FA oxidation during heavy exercise is distributed between ACS, CPT1, and ETC.

Quantitative and qualitative adaptation of human skeletal muscle mitochondria to hypoxic compared with normoxic training at the same relative work rate.

AIM: To investigate if training during hypoxia (H) improves the adaptation of muscle oxidative function compared with normoxic (N) training performed at the same relative intensity. METHOD: Eight untrained volunteers performed one-legged cycle training during 4 weeks in a low-pressure chamber. One leg was trained under N conditions and the other leg under hypobaric hypoxia (526 mmHg) at the same relative intensity as during N (65% of maximal power output, W(max)). Muscle biopsies were taken from vastus lateralis before and after the training period. Muscle samples were analysed for the activities of oxidative enzymes [citrate synthase (CS) and cytochrome c oxidase (COX)] and mitochondrial respiratory function. RESULTS: W(max) increased with more than 30% over the training period during both N and H. CS activity increased significantly after training during N conditions (+20.8%, P < 0.05) but remained unchanged after H training (+4.5%, ns) with a significant difference between conditions (P < 0.05 H vs. N). COX activity was not significantly changed by training and was not different between exercise conditions [+14.6 (N) vs. -2.3% (H), ns]. Maximal ADP stimulated respiration (state 3) expressed per weight of muscle tended to increase after N (+31.2%, P < 0.08) but not after H training (+3.2%, ns). No changes were found in state four respiration, respiratory control index, P/O ratio, mitochondrial Ca(2+) resistance and apparent Km for oxygen. CONCLUSION: The training-induced increase in muscle oxidative function observed during N was abolished during H. Altitude training may thus be disadvantageous for adaptation of muscle oxidative function.

Increased concentrations of P(i) and lactic acid reduce creatine-stimulated respiration in muscle fibers.

We tested the hypothesis that the respiratory function of skeletal muscle mitochondria is impaired by lactic acidosis and elevated concentrations of P(i). The rate of respiration of chemically skinned fiber bundles from rat soleus muscle was measured at [P(i)] (brackets denote concentration) and pH values similar to those at rest (3 mM P(i), pH 7.0) and high-intensity exercise (20 mM P(i), pH 6.6). Respiration was measured in the absence of ADP and after sequential additions of 0.1 mM ADP, 20 mM creatine (Cr; V(Cr)), and 4 mM ADP. Respiration at 0.1 mM ADP increased after addition of Cr. However, V(Cr) was 23% lower (P < 0.05) during high-intensity conditions than during resting conditions. V(Cr) was also reduced when P(i) or H(+) was increased separately (P < 0.05). Respiration in the absence of ADP and after additions of 0.1 mM ADP and 4 mM ADP was not affected by changes in [P(i)] or [H(+)]. The response was similar, irrespective of when acidosis was induced (i.e., quiescent or actively respiring mitochondria). In conclusion, Cr-stimulated respiration is impaired by increases in [H(+)] and [P(i)] corresponding to those in exercising muscle. Although the reduced Cr-stimulated respiration could be compensated for by increased [ADP], this might have implications for intracellular homeostasis.

The role of phosphorylcreatine and creatine in the regulation of mitochondrial respiration in human skeletal muscle.

1. The role of phosphorylcreatine (PCr) and creatine (Cr) in the regulation of mitochondrial respiration was investigated in permeabilised fibre bundles prepared from human vastus lateralis muscle. 2. Fibre respiration was measured in the absence of ADP (V(0)) and after sequential additions of submaximal ADP (0.1 mM ADP, V(submax)), PCr (or Cr) and saturating [ADP] (V(max)). 3. V(submax) increased by 55 % after addition of saturating creatine (P < 0.01; n = 8) and half the maximal effect was obtained at 5 mM [Cr]. In contrast, V(submax) decreased by 54 % after addition of saturating phosphorylcreatine (P < 0.01; n = 8) and half the maximal effect was obtained at 1 mM [PCr]. V(max) was not affected by Cr or PCr. 4. V(submax) was similar when PCr and Cr were added simultaneously at concentrations similar to those in muscle at rest (PCr/Cr = 2) and at low-intensity exercise (PCr/Cr = 0.5). At conditions mimicking high-intensity exercise (PCr/Cr = 0.1), V(submax) increased to 60 % of V(max) (P < 0.01 vs. rest and low-intensity exercise). 5. Eight of the subjects participated in a 16 day Cr supplementation programme. Following Cr supplementation, V(0) decreased by 17 % (P < 0.01 vs. prior to Cr supplementation), whereas ADP-stimulated respiration (with and without Cr or PCr) was unchanged. 6. For the first time evidence is given that PCr is an important regulator of mitochondrial ADP-stimulated respiration. Phosphorylcreatine decreases the sensitivity of mitochondrial respiration to ADP whereas Cr has the opposite effect. During transition from rest to high-intensity exercise, decreases in the PCr/Cr ratio will effectively increase the sensitivity of mitochondrial respiration to ADP. The decrease in V(0) after Cr supplementation indicates that intrinsic changes in membrane proton conductance occur.

Effect of endurance training on oxidative and antioxidative function in human permeabilized muscle fibres.

The adaptation of muscle oxidative function to 6 weeks of endurance cycle training was investigated in eight untrained subjects. Peak oxygen consumption (VO2peak) increased by 24% (2.69+/-0.21 versus 3.34+/-0.30 l O2 min(-1), mean +/-SEM, P<0.01) and lactate threshold intensity increased by 53% (121+/-13 versus 185+/-15 W, P<0.01) following the training period. Muscle biopsy samples were taken from vastus lateralis before and after training, and respiration in permeabilized muscle fibres was measured. Following training, non-ADP-stimulated respiration (VO) of skinned fibres increased by 35% (0.17+/-0.01 versus 0.23+/-0.01 mmol O2.min(-1).kg(-1) wet weight, P<0.05) and maximal ADP-stimulated respiration (VmaX) increased by 38% (1.17+/-0.07 versus 1.62+/-0.14 mmol O2.min(-1).kg(-1) wet weight, P<0.05). ADP sensitivity [i.e. the ratio between mitochondrial respiration (after correction for VO) at 0.1 mM ADP and Vmax] was reduced after training (0.40+/-0.05 versus 0.26+/-0.02; P<0.05). Mitochondrial resistance to oxidative stress was investigated by exposing skinned fibres to exogenous reactive oxygen species (ROS). ADP-stimulated respiration was reduced after ROS exposure and the relative decrease was similar before and after training. It is concluded that after endurance training: (1) the relative increase in maximal muscle fibre respiration exceeds that of whole-body oxygen uptake; (2) the sensitivity of mitochondrial respiration to ADP decreases; and (3) the impairment of oxidative function in skinned muscle fibres by ROS remains unchanged.

Effect of eccentric exercise on muscle oxidative metabolism in humans.

PURPOSE: The purpose of this study was to evaluate the effects of eccentric exercise on muscle oxidative function. METHODS: Thirteen subjects performed high-intensity eccentric cycling for 30 min. Muscle oxidative function in vastus lateralis was evaluated by measurements of respiration in permeabilized muscle fibers (skinned fibers) and from the kinetics of oxyhemoglobin (oxyHb) saturation measured with near infrared spectroscopy (NIRS). RESULTS: After eccentric cycling, all subjects reported extensive delayed onset muscle soreness (DOMS), but plasma markers of muscle damage (creatine kinase and beta-glucuronidase activity) were not significantly altered. The half time of oxyHb desaturation after circulatory occlusion (128 +/- 11 s, mean +/- SE) and oxyHb resaturation after restoration of blood flow (13.8 +/- 0.7 s) were not significantly changed after eccentric cycling (N = 7). Respiration in skinned muscle fibers measured in the absence of ADP and in the presence of a submaximal (0.1 mM) or maximal ADP concentration (1 mM) was not significantly changed after eccentric cycling (N = 6). The sensitivity of respiration to ADP was not significantly changed after eccentric cycling. CONCLUSIONS: Muscle oxidative function (maximal respiration and respiratory control by ADP) was not compromised after high-intensity eccentric cycle exercise. Furthermore, NIRS indicates that after eccentric cycling muscle oxygen utilization and local oxygen transport at rest are unchanged. It is concluded that eccentric cycling, although causing DOMS, does not negatively affect skeletal muscle oxidative function.

Functional complexes of mitochondria with Ca,MgATPases of myofibrils and sarcoplasmic reticulum in muscle cells.

Regulation of mitochondrial respiration in situ in the muscle cells was studied by using fully permeabilized muscle fibers and cardiomyocytes. The results show that the kinetics of regulation of mitochondrial respiration in situ by exogenous ADP are very different from the kinetics of its regulation by endogenous ADP. In cardiac and m. soleus fibers apparent K(m) for exogenous ADP in regulation of respiration was equal to 300-400 microM. However, when ADP production was initiated by intracellular ATPase reactions, the ADP concentration in the medium leveled off at about 40 microM when about 70% of maximal rate of respiration was achieved. Respiration rate maintained by intracellular ATPases was suppressed about 20-30% during exogenous trapping of ADP with excess pyruvate kinase (PK, 20 IU/ml) and phosphoenolpyruvate (PEP, 5 mM). ADP flux via the external PK+PEP system was decreased by half by activation of mitochondrial oxidative phosphorylation. Creatine (20 mM) further activated the respiration in the presence of PK+PEP. It is concluded that in oxidative muscle cells mitochondria behave as if they were incorporated into functional complexes with adjacent ADP producing systems - with the MgATPases in myofibrils and Ca,MgATPases of sarcoplasmic reticulum.

Endurance training increases stimulation of uncoupling of skeletal muscle mitochondria in humans by non-esterified fatty acids: an uncoupling-protein-mediated effect?

Uncoupled respiration (UCR) is an essential property of muscle mitochondria and has several functions in the cell. We hypothesized that endurance training may alter the magnitude and properties of UCR in human muscle. Isolated mitochondria from muscle biopsies taken before and after 6 weeks of endurance exercise training (n=8) were analysed for UCR. To investigate the role of uncoupling protein 2 (UCP2) and UCP3 in UCR, the sensitivity of UCR to UCP-regulating ligands (non-esterified fatty acids and purine nucleotides) and UCP2 and UCP3 mRNA expression in muscle were examined. Oleate increased the mitochondrial oxygen consumption rate, an effect that was not attenuated by GDP and/or cyclosporin A. The effect of oleate was significantly greater after compared with before training. Training had no effect on UCP2 or UCP3 mRNA levels, but after training the relative increase in respiration rate induced by oleate was positively correlated with the UCP2 mRNA level. In conclusion, we show that the sensitivity of UCR to non-esterified fatty acids is up-regulated by endurance training. This suggests that endurance training causes intrinsic changes in mitochondrial function, which may enhance the potential for regulation of aerobic energy production, prevent excess free radical generation and contribute to a higher basal metabolic rate.

Mitochondrial function and antioxidative defence in human muscle: effects of endurance training and oxidative stress.

The influence of endurance training on oxidative phosphorylation and the susceptibility of mitochondrial oxidative function to reactive oxygen species (ROS) was investigated in skeletal muscle of four men and four women. Mitochondria were isolated from muscle biopsies taken before and after 6 weeks of endurance training. Mitochondrial respiration was measured before and after exposure of mitochondria to exogenous ROS (H2O2 + FeCl2). Endurance training increased peak pulmonary O2 uptake (VO2,peak) by 24 % and maximal ADP-stimulated mitochondrial oxygen consumption (state 3) by 40% (P<0.05). Respiration in the absence of ADP (state 4), the respiratory control ratio (RCR = state 3/state 4) and the ratio between added ADP and consumed oxygen (P/O) remained unchanged by the training programme. Exposure to ROS reduced state 3 respiration but the effect was not significantly different between pre- and post-training samples. State 4 oxygen consumption increased after exposure to ROS both before (+189 %, P< 0.05) and after training (+243 %, P<0.05) and the effect was significantly higher after training (P<0.05, pre- vs. post-training). The augmented state 4 respiration could in part be attenuated by atractyloside, which indicates that ADP/ATP translocase was affected by ROS. The P/O ratio in ROS-treated mitochondria was significantly lower (P<0.05) compared to control conditions, both before (-18.6+/-2.2 %) and after training (-18.5+/-1.1%). Muscle activities of superoxide dismutase (mitochondrial and cytosolic), glutathione peroxidase and muscle glutathione status were unaffected by training. There was a positive correlation between muscle superoxide dismutase activity and age (r = 0.75; P<0.05; range of age 20-37 years), which may reflect an adaptation to increased generation of ROS in senescent muscle. The muscle glutathione pool was more reduced in subjects with high activity of glutathione peroxidase (r = 0.81; P<0.05). The influence of short-term training on mitochondrial oxygen consumption has for the first time been investigated in human skeletal muscle. The results showed that maximal mitochondrial oxidative power is increased after endurance training but that the efficiency of energy transfer (P/O ratio) remained unchanged. Antioxidative defence was unchanged after training when expressed relative to muscle weight. Although this corresponds to a reduced antioxidant protection per individual mitochondrion, the sensitivity of aerobic energy transfer to ROS was unchanged. However, the augmented ROS-induced non-coupled respiration after training indicates an increased susceptibility of mitochondrial membrane proton conductance to oxidative stress.

Plasma hypoxanthine and ammonia in humans during prolonged exercise.

In this study we examined the time course of changes in the plasma concentration of oxypurines [hypoxanthine (Hx), xanthine and urate] during prolonged cycling to fatigue. Ten subjects with an estimated maximum oxygen uptake (VO2(max)) of 54 (range 47-67) ml x kg(-1) x min(-1) cycled at [mean (SEM)] 74 (2)% of VO2(max) until fatigue [79 (8) min]. Plasma levels of oxypurines increased during exercise, but the magnitude and the time course varied considerably between subjects. The plasma concentration of Hx ([Hx]) was 1.3 (0.3) micromol/l at rest and increased eight fold at fatigue. After 60 min of exercise plasma [Hx] was >10 micromol/l in four subjects, whereas in the remaining five subjects it was <5 micromol/l. The muscle contents of total adenine nucleotides (TAN = ATP+ADP+AMP) and inosine monophosphate (IMP) were measured before and after exercise in five subjects. Subjects with a high plasma [Hx] at fatigue also demonstrated a pronounced decrease in muscle TAN and increase in IMP. Plasma [Hx] after 60 min of exercise correlated significantly with plasma concentration of ammonia ([NH(3)], r = 0.90) and blood lactate (r = 0.66). Endurance, measured as time to fatigue, was inversely correlated to plasma [Hx] at 60 min (r = -0.68, P < 0.05) but not to either plasma [NH(3)] or blood lactate. It is concluded that during moderate-intensity exercise, plasma [Hx] increases, but to a variable extent between subjects. The present data suggest that plasma [Hx] is a marker of adenine nucleotide degradation and energetic stress during exercise. The potential use of plasma [Hx] to assess training status and to identify overtraining deserves further attention.

Actively phosphorylating mitochondria are more resistant to lactic acidosis than inactive mitochondria.

Oxidative phosphorylation of isolated rat skeletal muscle mitochondria after exposure to lactic acidosis in either phosphorylating or nonphosphorylating states has been evaluated. Mitochondrial respiration and transmembrane potential (DeltaPsi(m)) were measured with pyruvate and malate as the substrates. The addition of lactic acid decreased the pH of the reaction medium from 7.5 to 6.4. When lactic acid was added to nonphosphorylating mitochondria, the subsequent maximal ADP-stimulated respiration decreased by 27% compared with that under control conditions (P < 0.05), and the apparent Michaelis-Menten constant (K(m)) for ADP decreased to 10 microM vs. 20 microM (P < 0.05) in controls. In contrast, maximal respiration and ADP sensitivity were not affected when mitochondria were exposed to acidosis during active phosphorylation in state 3. Acidosis significantly increased mitochondrial oxygen consumption in state 4 (post-state 3), irrespective of when acidosis was induced. This effect of acidosis was attenuated in the presence of oligomycin. The addition of lactic acid during state 4 respiration decreased DeltaPsi(m) by 19%. The ratio between added ADP and consumed oxygen (P/O) was close to the theoretical value of 3 in all conditions. The addition of potassium lactate during state 3 (i.e., medium pH unchanged) had no effect on the parameters measured. It is concluded that lactic acidosis has different effects when induced on nonphosphorylating vs. actively phosphorylating mitochondria. On the basis of these results, we suggest that the influence of lactic acidosis on muscle aerobic energy production depends on the physiological conditions at the onset of acidity.

Effect of Q10 supplementation on tissue Q10 levels and adenine nucleotide catabolism during high-intensity exercise.

The aim of the present study was to investigate the concentration of ubiquinone-10 (Q10), at rest, in human skeletal muscle and blood plasma before and after a period of high-intensity training with or without Q10 supplementation. Another aim was to explore whether adenine nucleotide catabolism, lipid peroxidation, and mitochondrial function were affected by Q10 treatment. Seventeen young healthy men were assigned to either a control (placebo) or Q10-supplementation (120 mg/day) group. Q10 supplementation resulted in a significantly higher plasma Q10/total cholesterol level on Days 11 and 20 compared with Day 1. There was no significant change in the concentration of Q10 in skeletal muscle or in isolated skeletal muscle mitochondria in either group. Plasma hypoxanthine and uric acid concentrations increased markedly after each exercise test session in both groups. After the training period, the postexercise increase in plasma hypoxanthine was markedly reduced in both groups, but the response was partially reversed after the recovery period. It was concluded that Q10 supplementation increases the concentration of Q10 in plasma but not in skeletal muscle.

Mitochondrial function in human skeletal muscle is not impaired by high intensity exercise.

The hypothesis that high-intensity (HI) intermittent exercise impairs mitochondrial function was investigated with different microtechniques in human muscle samples. Ten male students performed three bouts of cycling at 130% of peak O2 consumption (V.O2,peak). Muscle biopsies were taken from the vastus lateralis muscle at rest, at fatigue and after 110 min recovery. Mitochondrial function was measured both in isolated mitochondria and in muscle fibre bundles made permeable with saponin (skinned fibres). In isolated mitochondria there was no change in maximal respiration, rate of adenosine 5'-triphosphate (ATP) production (measured with bioluminescence) and respiratory control index after exercise or after recovery. The ATP production per consumed oxygen (P/O ratio) also remained unchanged at fatigue but decreased by 4% (P<0.05) after recovery. In skinned fibres, maximal adenosine 5'-diphosphate (ADP)-stimulated respiration increased by 23% from rest to exhaustion (P<0.05) and remained elevated after recovery, whereas the respiratory rates in the absence of ADP and at 0.1 mM ADP (submaximal respiration) were unchanged. The ratio between respiration at 0.1 and 1 mM ADP (ADP sensitivity index) decreased at fatigue (P<0.05) but after the recovery period was not significantly different from that at rest. It is concluded that mitochondrial oxidative potential is maintained or improved during exhaustive HI exercise. The finding that the sensitivity of mitochondrial respiration to ADP is reversibly decreased after strenuous exercise may indicate that the control of mitochondrial respiration is altered.

Mitochondrial oxidative function in human saponin-skinned muscle fibres: effects of prolonged exercise.

1. The influence of prolonged exhaustive exercise on mitochondrial oxidative function was investigated in ten men. 2. Muscle biopsies were taken before and after exercise and mitochondrial respiration investigated in fibre bundles made permeable by pretreatment with saponin. 3. After exercise, respiration in the absence of ADP increased by 18 % (P < 0.01), but respiration at suboptimal ADP concentration (0.1 mM) and maximal ADP-stimulated respiration (1 mM ADP) remained unchanged. 4. In the presence of creatine (20 mM), mitochondrial affinity for ADP increased markedly and respiration at suboptimal ADP concentration (0.1 mM) was similar (pre-exercise) or higher (post-exercise; P < 0.05) than with 1 mM ADP alone. The increase in respiratory rate with creatine was correlated to the relative type I fibre area (r = 0.84). Creatine-stimulated respiration increased after prolonged exercise (P < 0.01). 5. The respiratory control index (6.8 +/- 0.4, mean +/- s.e.m.) and the ratio between respiration at 0.1 and 1 mM ADP (ADP sensitivity index, 0.63 +/- 0.03) were not changed after exercise. The sensitivity index was negatively correlated to the relative type I fibre area (r = -0.86). 6. The influence of exercise on muscle oxidative function has for the first time been investigated with the skinned-fibre technique. It is concluded that maximal mitochondrial oxidative power is intact or improved after prolonged exercise, while uncoupled respiration is increased. The latter finding may contribute to the elevated post-exercise oxygen consumption. The finding that the sensitivity of mitochondrial respiration for ADP and creatine are related to fibre-type composition indicates intrinsic differences in the control of mitochondrial respiration between fibres.

Energy supply and muscle fatigue in humans.

Limitations in energy supply is a classical hypothesis of muscle fatigue. The present paper reviews the evidence available from human studies that energy deficiency is an important factor in fatigue. The maximal rate of energy expenditure determined in skinned fibres is close to the rate of adenosine triphosphate (ATP) utilisation observed in vivo and data suggest that performance during short bursts of exercise (<5 s duration) primarily is limited by other factors than energy supply (e.g. Vmax of myosine adenosine triphosphatase (ATPase), motor unit recruitment, engaged muscle mass). Within 10 s of exercise maximal power output decreases considerably and coincides with depletion of phosphocreatine. During recovery, maximal force and power output is restored with a similar time course as the resynthesis of phosphocreatine. Increases in muscle store of phosphocreatine through dietary supplementation with creatine increases performance during high-intensity exercise. These findings support the hypothesis that energy supply limits performance during high-intensity exercise. It is well documented that pre-exercise muscle glycogen content is related to performance during moderate intensity exercise. Recent data indicates that the interfibre variation in phosphocreatine is large after prolonged exercise to fatigue and that some fibres are depleted to the same extent as after high-intensity exercise. Despite relatively small decreases in ATP, the products of ATP hydrolysis (Pi and free ADP) may increase considerably. Free ADP calculated from the creatine kinase reaction increases 10-fold both after high-intensity exercise and after prolonged exercise to fatigue. It is suggested that local increases in ADP may reach inhibitory levels for the contraction process.

Rate of oxidative phosphorylation in isolated mitochondria from human skeletal muscle: effect of training status.

Muscle oxidative function has been investigated in subjects with various training status (VO2 max, 41-72 mL O2 kg-1 body wt min-1, n = 10). Mitochondria were isolated from biopsies taken from m. vastus lateralis. Maximal mitochondrial oxygen consumption (QO2) and ATP production (MAPR) were measured with polarographic and bioluminometric techniques, respectively. The yield of mitochondria, calculated from the fractional activity of citrate synthase (CS), averaged 26%. With pyruvate + malate, the respiratory control ratio was 5.7 +/- 0.4 (X +/- SE) and the P/O ratio was 2.83 +/- 0.02, which demonstrates that the isolated mitochondria were functionally intact. QO2 was significantly correlated to aerobic training status expressed as muscle CS activity (r = 0.86), VO2 max (r = 0.84) and lactate threshold (r = 0.83) but not to the fibre type composition. A highly significant correlation (r = 0.93) was observed between ATP production calculated from QO2 and MAPR, but ATP production derived from QO2 was higher than MAPR both for pyruvate + malate (255%) and for alpha-ketoglutarate (23%). QO2 extrapolated to a temperature of 38 degrees C averaged 68 mL O2 min-1 kg-1 wet wt, which is similar to previous findings in vitro and in vivo during the post-exercise period. However, calculated muscle O2 utilization during exercise was three- to fivefold higher than QO2 measured on isolated mitochondria. It is suggested that additional factors exist for activation of mitochondrial respiration during exercise. It is concluded that muscle oxidative function can be quantitatively assessed from the respiration of mitochondria isolated from needle biopsy specimens and that QO2 is closely correlated to whole-body VO2 max.

Phosphocreatine content in single fibers of human muscle after sustained submaximal exercise.

The effect of sustained submaximal exercise on muscle energetics has been studied on the single-fiber level in human skeletal muscle. Seven subjects cycled to fatigue (mean 77 min) at a work rate corresponding to approximately 75% of maximal O2 uptake. Biopsies were taken from the vastus lateralis muscle at rest, at fatigue, and after 5 min of recovery. Muscle glycogen decreased from 444 +/- 40 (SE) mmol glucosyl units/kg dry wt at rest to 94 +/- 16. Postexercise glycogen was inversely correlated (P < 0.01) to muscle content of inosine monophosphate, a catabolite of ATP. Phosphocreatine (PCr) in mixed-fiber muscle decreased at fatigue to 37% but was restored above the initial value (106.5%, P < 0.025) after 5 min of recovery. The overshoot was localized to type I fibers. The rapid reversal of PCr is in contrast to the slow recovery in contraction force. Pi increased at fatigue but less than that expected from the changes in PCr and other phosphate compounds. Mean PCr at rest was approximately 20% higher in type II than in type I fibers (86.4 +/- 3.6 and 71.6 +/- 1.8 mmol/kg dry wt, respectively, P < 0.05), but at fatigue similar PCr contents were observed in the two fiber types. Reduction in PCr in all fibers at fatigue suggests that all fibers were recruited at the end of exercise. PCr content in single fibers showed a great variability in samples at rest, exercise, and recovery. The variability was more pronounced than for ATP, and the data suggest that it is due to interfiber physiological-biochemical differences. At fatigue ATP was maintained relatively high in all single fibers, but a pronounced depletion of PCr was observed in a large number of fibers, and this may contribute to fatigue through the associated increases in Pi or/and free ADP. It is noteworthy that the increase in calculated free ADP at fatigue was similar to that after high-intensity exercise.