Sex differences in motor unit behaviour: A review.

The lack of systematic investigations on sex-related differences in motor unit behaviour poses a challenge in understanding and optimizing health and performance in males and females. Limited investigations revealed that sex differences in motor unit behaviour might be present in human muscles. This review summarizes the current knowledge on sex differences in motor unit behaviour and potential factors that may contribute to these differences. We show significant under-representation of female participants in motor unit studies and a limited number of studies investigating sex differences in motor unit behaviour. We place the current insights within the context of methodological limitations and outline several recommendations and future directions to improve female representation in this research area. We conclude that there is an urgent need to gather more data in females and investigate sex differences in motor unit behaviour. The knowledge gained could be used to develop sex-specific approaches to improve neuromuscular performance and rehabilitation.

Sex differences in the modulation of the motor unit discharge rate leads to reduced force steadiness.

The purpose of this study was to evaluate the relationship between the variability in the motor unit inter-pulse interval and force steadiness at submaximal and maximal force outputs between the sexes. Twenty-four male and 24 female participants were recruited to perform isometric dorsiflexion contractions at 20, 40, 60, 80, and 100% maximum voluntary contraction. Tibialis anterior myoelectric signal was recorded by an intramuscular electrode. Females had lower force steadiness (coefficient of variation of force (CoV-Force), 27.3%, p < 0.01) and a greater coefficient of variation of motor unit action potential inter-pulse interval (CoV-IPI), compared with males (9.6%, p < 0.01). There was no significant correlation between the normalized CoV-IPI and CoV-Force (r = 0.19, p > 0.01), but there was a significant repeated measures correlation between the raw scores for root-mean-square force error and the standard deviation of motor unit discharge rate (r = 0.65, p < 0.01). Females also had a greater incidence of doublet discharges on average across force levels (p < 0.01). The sex differences may result from motor unit behaviours (i.e., doublet and rapid discharges, synchronization, rate coding or recruitment), leading to lower force steadiness and greater CoV-IPI in females. Novelty: Sex differences in force steadiness may be due to neural strategies. Females have lower force steadiness compared with males. Greater incidence of doublet discharges in females may result in lesser force steadiness.

Is the 'reverse onion skin' phenomenon more prevalent than we thought during intramuscular myoelectric recordings from low to maximal force outputs?

There are isolated instances in the literature that suggest the 'onion skin' phenomenon is not always present. That is, newly recruited high threshold motor units (MU) have higher discharge rates than previously recruited low threshold MUs. Therefore, the purpose of this paper was to investigate the presence of the 'onion skin' phenomenon in a large sample of intramuscular myoelectric recordings from low to maximal force outputs. Forty-eight participants performed rapid isometric dorsiflexion contractions at 20, 40, 60, 80 and 100 % MVC while intramuscular electrical activity was recorded. A bivariate frequency-distribution of the motor unit discharge rate and motor unit action potential peak-to-peak (P-P) amplitude was assessed. There was a significant difference in bivariate frequency-distribution across force levels (D's = 0.1083-0.3094, p's < 0.001). Newly recruited high threshold MUs did have lower discharge rates, but there was an increase in the presence of high threshold, large P-P amplitude MUs with higher discharge rates than lower threshold MUs (reverse onion skin) during the stable portion of the force output. The recruitment of high threshold MUs with higher discharge rates decreased the level of common drive from the cross-correlation (Rxy) = 0.79 at 20 % MVC to Rxy = 0.68 at 100 % MVC (p < 0.01), but it remained high. As the interference pattern becomes more complex with the recruitment of more motor units at higher force outputs, intramuscular electrodes may be more discriminating while recording motor unit activity leading to the identification of both the 'reverse onion skin' and 'onion skin' phenomenon being present.

Sex differences in motor unit discharge rates at maximal and submaximal levels of force output.

This study evaluated potential sex differences in motor unit (MU) behaviour at maximal and submaximal force outputs. Forty-eight participants, 24 females and 24 males, performed isometric dorsiflexion contractions at 20%, 40%, 60%, 80%, and 100% of a maximum voluntary contraction (MVC). Tibialis anterior electromyography was recorded both by surface and intramuscular electrodes. Compared with males, females had a greater MU discharge rate (MUDR) averaged across all submaximal intensities (Δ 0.45 pps, 2.56%). Males exhibited greater increases in MUDR above 40% MVC, surpassing females at 100% MVC (p's < 0.01). Averaged across all force outputs, females had a greater incidence of doublet and rapid discharges and a greater percentage of MU trains with doublet and rapid (5-10 ms) discharges (Δ 75.55% and 61.48%, respectively; p's < 0.01). A subset of males (n = 8) and females (n = 8), matched for maximum force output, revealed that females had even greater MUDR (Δ 1.38 pps, 7.47%) and percentage of MU trains with doublet and rapid discharges (Δ 51.62%, 56.68%, respectively; p's < 0.01) compared with males at each force output, including 100% MVC. Analysis of the subset of strength-matched males and females suggest that sex differences in MU behaviour may be a result of females needing to generate greater neural drive to achieve fused tetanus. Novelty Females had higher MUDRs and greater percentage of MU trains with doublets across submaximal force outputs (20%-80% MVC). Differences were even greater for a strength matched subset. Differences in motor unit behaviour may arise from musculoskeletal differences, requiring greater neural drive in females.

Neural, biomechanical, and physiological factors involved in sex-related differences in the maximal rate of isometric torque development.

OBJECTIVE: Recent research has reported that lower maximal rate of torque development (dτ/dt max) exhibited by females, relative to males, during knee extension can be accounted for by normalization to a maximal voluntary contraction (MVC); however, this was not seen in the upper limb. PURPOSE: The aim of the current work was to examine the contribution of maximum strength (τmax), twitch contraction time (CT), muscle fiber condition velocity (MFCV), and rate of muscle activation (Q30) to sex-differences in the dτ/dt max during maximal isometric dorsiflexion. METHODS: Thirty-eight participants (20 males; 18 females) performed both maximal voluntary and evoked isometric contractions of the tibialis anterior across 3 days. Ten maximal compound muscle action potentials were elicited and subsequently followed by three, 5-s contractions. From the recordings, MFCV, dτ/dt max, τmax, CT, electromechanical delay (EMD), root-mean squared (RMS) amplitude, peak-to-peak voltage (Vpp), and Q30 were calculated. RESULTS: An ANCOVA showed that τmax accounted for all the sex-differences in dτ/dt max (p = 0.96). There were no significant differences between groups with respect to MFCV, RMS amplitude, Vpp amplitude, or CT. However, there was a significant sex-difference in dτ/dt max, τmax, and Q30. Females had longer evoked EMD times compared with males (15.69 ± 10.57 ms versus 9.95 ± 3.46 ms; p = 0.01), but the voluntary EMD times were not different. CONCLUSION: The current research supports the work by Hannah et al. Exp Physiol 97:618-629, (2012) that normalization to MVC in the quadriceps is able to account for all sex-differences in rate of toque development in the lower limb.

Elevated concentrations of circulating cytokines and correlations with nerve conduction velocity in human peripheral nerves.

The purpose of the current study was to quantify the potential relationship between various cytokines and peripheral nerve function in humans, in-vivo. Measures of nerve conduction velocity (NCV) were examined prior to and following the induction of a cytokine spike. A significant negative correlation was found between the change in IL-1ra and the change in NCV at 24h post-exercise (r=-0.65, p=0.02) while a significant positive correlation was found between the change in IL-6 and the change in NCV at 2h post-exercise (r=0.61, p=0.048). It may be possible that different cytokines induce a unique neural influence at elevated concentrations.

Sex-related differences in maximal rate of isometric torque development.

Sex-differences in the maximum rate of torque development (dτ/dt(max)) may be due to differences in maximum muscle strength, because higher torque values mathematically lead to higher values for the rate of change in torque. The rate of change in the isometric torque-time curve is often normalized to the isometric maximum voluntary contraction (MVC) to evaluate males and females on a relative scale. Normalization eliminates sex-differences in dτ/dt(max) in the lower limbs because males and females are more comparable (i.e., differences between the sexes are relatively small) with respect to both muscle size and strength. However, normalization fails to result in parody in dτ/dt(max) of the upper limb, leading to the idea that other factors may be involved. This study determined if sex-differences in dτ/dt(max) in the upper limb can be attributed to differences in isometric MVC and/or a neural variable related to rate of increase in muscle activation (Q(30)). Forty-six participants (23 males, 23 females) performed maximal isometric elbow flexion contractions, "as hard and as fast as possible". Maximum torque (τ(max)), dτ/dt(max), and the rate of increase in surface electromyographic (sEMG) activity (Q(30)) were assessed. Muscle plus bone cross-sectional area (M+B CSA) of the upper arm was calculated to estimate differences in muscle size, only for comparative purposes. Maximum strength (55.5%) and muscle size (41.9%) of the elbow flexors in males were much greater than that of females (p < 0.05). There was a large difference (61.2%) between males and females with respect to dτ/dt(max) that was reduced by statistical correction using an analysis of covariance (ANCOVA). The percent differences were reduced to 36.7% (p < 0.05) for τ(max) and 54.4% (p < 0.05) for Q30, but was nearly eliminated to 13.8% (p > 0.05) when both variables were used simultaneously as covariates. Since sex-differences in the upper limb dτ/dt(max) persist, additional neural or biomechanical factors may be involved.

The relationship between human skeletal muscle pyruvate dehydrogenase phosphatase activity and muscle aerobic capacity.

Pyruvate dehydrogenase (PDH) is a mitochondrial enzyme responsible for regulating the conversion of pyruvate to acetyl-CoA for use in the tricarboxylic acid cycle. PDH is regulated through phosphorylation and inactivation by PDH kinase (PDK) and dephosphorylation and activation by PDH phosphatase (PDP). The effect of endurance training on PDK in humans has been investigated; however, to date no study has examined the effect of endurance training on PDP in humans. Therefore, the purpose of this study was to examine differences in PDP activity and PDP1 protein content in human skeletal muscle across a range of muscle aerobic capacities. This association is important as higher PDP activity and protein content will allow for increased activation of PDH, and carbohydrate oxidation. The main findings of this study were that 1) PDP activity (r(2) = 0.399, P = 0.001) and PDP1 protein expression (r(2) = 0.153, P = 0.039) were positively correlated with citrate synthase (CS) activity as a marker for muscle aerobic capacity; 2) E1α (r(2) = 0.310, P = 0.002) and PDK2 protein (r(2) = 0.229, P =0.012) are positively correlated with muscle CS activity; and 3) although it is the most abundant isoform, PDP1 protein content only explained ∼ 18% of the variance in PDP activity (r(2) = 0.184, P = 0.033). In addition, PDP1 in combination with E1α explained ∼ 38% of the variance in PDP activity (r(2) = 0.383, P = 0.005), suggesting that there may be alternative regulatory mechanisms of this enzyme other than protein content. These data suggest that with higher muscle aerobic capacity (CS activity) there is a greater capacity for carbohydrate oxidation (E1α), in concert with higher potential for PDH activation (PDP activity).

PDH activation during in vitro muscle contractions in PDH kinase 2 knockout mice: effect of PDH kinase 1 compensation.

Pyruvate dehydrogenase (PDH) plays an important role in regulating carbohydrate oxidation in skeletal muscle. PDH is deactivated by a set of PDH kinases (PDK1, PDK2, PDK3, PDK4), with PDK2 and PDK4 being the most predominant isoforms in skeletal muscle. Although PDK2 is the most abundant isoform, few studies have examined its physiological role. The role of PDK2 on PDH activation (PDHa) at rest and during muscle stimulation at 10 and 40 Hz (eliciting low- and moderate-intensity muscle contractions, respectively) in isolated extensor digitorum longus muscles was studied in PDK2 knockout (PDK2KO) and wild-type (WT) mice (n = 5 per group). PDHa activity was unexpectedly 35 and 77% lower in PDK2KO than WT muscle (P = 0.043), while total PDK activity was nearly fourfold lower in PDK2KO muscle (P = 0.006). During 40-Hz contractions, initial force was lower in PDK2KO than WT muscle (P < 0.001) but fatigued similarly to ∼75% of initial force by 3 min. There were no differences in initial force or rate of fatigue during 10-Hz contractions. PDK1 compensated for the lack of PDK2 and was 1.8-fold higher in PDK2KO than WT muscle (P = 0.019). This likely contributed to ensuring that resting PDHa activity was similar between the groups and accounts for the lower PDH activation during muscle contraction, as PDK1 is a very potent inhibitor of the PDH complex. Increased PDK1 expression appears to be regulated by hypoxia inducible factor-1α, which was 3.5-fold higher in PDK2KO muscle. It is clear that PDK2 activity is essential, even at rest, in regulation of carbohydrate oxidation and production of reducing equivalents for the electron transport chain. In addition, these results underscore the importance of the overall kinetics of the PDK isoform population, rather than total PDK activity, in determining transformation of the PDH complex and PDHa activity during muscle contraction.

Experimental and modelling investigation of surface EMG spike analysis.

A pattern classification method based on five measures extracted from the surface electromyographic (sEMG) signal is used to provide a unique characterization of the interference pattern for different motor unit behaviours. This study investigated the sensitivity of the five sEMG measures during the force gradation process. Tissue and electrode filtering effects were further evaluated using a sEMG model. Subjects (N=8) performed isometric elbow flexion contractions from 0 to 100% MVC. The sEMG signals from the biceps brachii were recorded simultaneously with force. The basic building block of the sEMG model was the detection of single fibre action potentials (SFAPs) through a homogeneous, equivalent isotropic, infinite volume conduction medium. The SFAPs were summed to generate single motor unit action potentials. The physiologic properties from a well-known muscle model and motor unit recruitment and firing rate schemes were combined to generate synthetic sEMG signals. The following pattern classification measures were calculated: mean spike amplitude, mean spike frequency, mean spike slope, mean spike duration, and the mean number of peaks per spike. Root-mean-square amplitude and mean power frequency were also calculated. Taken together, the experimental data and modelling analysis showed that below 50% MVC, the pattern classification measures were more sensitive to changes in force than traditional time and frequency measures. However, there are additional limitations associated with electrode distance from the source that must be explored further. Future experimental work should ensure that the inter-electrode distance is no greater than 1cm to mitigate the effects of tissue filtering.

Carbohydrate refeeding after a high-fat diet rapidly reverses the adaptive increase in human skeletal muscle PDH kinase activity.

Pyruvate dehydrogenase (PDH) regulates oxidative carbohydrate disposal in skeletal muscle and is downregulated by reversible phosphorylation catalyzed by PDH kinase (PDK). Previous work has demonstrated increased PDK activity and PDK4 expression in human skeletal muscle following a high-fat low-carbohydrate (HF) diet, which leads to decreased PDH in the active form (PDHa activity) and carbohydrate oxidation. The purpose of this study was to examine the time course of changes in PDK and PDHa activities with refeeding of carbohydrates after an HF diet in human skeletal muscle. Healthy male volunteers (n = 8) consumed a standardized 3-day Pre-diet with the same energy content as their habitual diet, followed by a eucaloric 6-day HF diet (Pre-diet: 50:30:20%; HF diet: 5:75:20%; carbohydrate/fat/protein). Muscle biopsies were taken before and after the HF diet and at 45 min and 3 h after carbohydrate refeeding with a single high-glycemic index carbohydrate meal (88:5:7% carbohydrate/fat/protein) representing approximately one third of the individual subject's habitual energy intake. PDK activity increased from 0.08 +/- 0.01 Pre- to 0.25 +/- 0.02 min (P < 0.001) Post-HF diet, and decreased with carbohydrate refeeding to 0.17 +/- 0.05 (P = 0.014) and 0.11 +/- 0.01 min (P = 0.006) at 45 min and 3 h, respectively. PDHa decreased from 0.89 +/- 0.20 to 0.32 +/- 0.05 (P = 0.007) mmol x min(-1) x kg wet wt(-1) following the HF diet, and was increased transiently with refeeding at 45 min, but returned to lower values by 3 h (P = 0.025 compared with Pre). The potential mechanism(s) for this attenuation of PDHa activity remains unclear. These data demonstrate that in human skeletal muscle, the adaptive increase in PDK activity following an HF diet is rapidly reversed to Pre-diet activity levels within 45 min to 3 h, and this is accompanied by a short-term increase in PDHa activity.

Skeletal muscle type comparison of pyruvate dehydrogenase phosphatase activity and isoform expression: effects of obesity and endurance training.

Pyruvate dehydrogenase (PDH) plays an important role in regulating carbohydrate metabolism in skeletal muscle. PDH is activated by PDH phosphatase (PDP) and deactivated by PDH kinase (PDK). Obesity has a large negative impact on skeletal muscle carbohydrate metabolism, whereas endurance training has been shown to improve regulatory control of skeletal muscle carbohydrate metabolism, more so when coupled with obesity. A majority of this literature has focused on PDK, with little information available on PDP. To determine the relative role of PDP in regulating skeletal muscle PDH activity with obesity and endurance training, obese and lean Zucker rats remained sedentary or were endurance trained (1 h/day, 5 days/wk) for a period of 8 wk. Soleus, red gastrocnemius, (RG), and white gastrocnemius (WG) muscles were sampled after the training period. The main findings were 1) obesity resulted in a 46% decrease in PDP activity expressed per milligram extracted mitochondrial protein only in RG, while PDP isoform content was unchanged; 2) 8 wk of endurance training led to a significant 1.4-2.2-fold increase in PDP activity of all muscle examined from obese rats, and the concomitant increase in PDP1 protein was only seen in soleus and RG; 3) 8 wk of endurance training led to a trending 1.4-2.2-fold increase in PDP activity of all muscle examined from obese rats, and the concomitant increase in PDP1 protein was only seen in soleus and RG; and 4) PDP2 protein content was not affected by obesity or training. These results suggest that decreased PDP activity in oxidative skeletal muscles may play a role in the impairment of carbohydrate metabolism in obese rats, which is reversible with endurance training.

The acute effects of differential dietary fatty acids on human skeletal muscle pyruvate dehydrogenase activity.

Pyruvate dehydrogenase (PDH) is an important regulator of carbohydrate oxidation during exercise, and its activity can be downregulated by an increase in dietary fat. The purpose of this study was to determine the acute metabolic effects of differential dietary fatty acids on the activation of the PDH complex (PDHa activity) at rest and at the onset of moderate-intensity exercise. University-aged male subjects (n = 7) underwent two fat-loading trials spaced at least 2 wk apart. Subjects consumed approximately 300 g saturated (SFA) or n-6 polyunsaturated fatty acid (PUFA) fat over the course of 5 h. Following this, participants cycled at 65% of their maximum oxygen uptake for 15 min. Muscle biopsies were taken before and following fat loading and at 1 min exercise. Plasma free fatty acids increased from 0.15 +/- 0.07 to 0.54 +/- 0.19 mM over 5 h with SFA and from 0.11 +/- 0.04 to 0.35 +/- 0.13 mM with n-6 PUFA and were significantly lower throughout the n-6 PUFA trial. PDHa activity was unchanged following fat loading but increased at the onset of exercise in the SFA trial, from 1.18 +/- 0.27 to 2.16 +/- 0.37 mmol x min(-1) x kg wet wt(-1). This effect was negated in the n-6 PUFA trial (1.04 +/- 0.20 to 1.28 +/- 0.36 mmol x min(-1) x kg wet wt(-1)). PDH kinase was unchanged in both trials, suggesting that the attenuation of PDHa activity with n-6 PUFA was a result of changes in the concentrations of intramitochondrial effectors, potentially intramitochondrial NADH or Ca(2+). Our findings suggest that attenuated PDHa activity contributes to the preferential oxidation of n-6 PUFA during moderate-intensity exercise.

Practice evoking the flexor carpi radialis h-reflex: a guideline for proficiency.

The purpose of this research was to identify the number of sessions required for a new investigator to become proficient at evoking an H-reflex in the flexor carpi radialis (FCR), in comparison to an experienced investigator. 31 students from Brock University in the greater Niagara region (16 women M age = 32.2, SD = 8.9 yr.; 15 men M age = 27.8, SD = 7.8 yr.) with no known neurological disorders volunteered and completed two test sessions performed by either an experienced or a novice investigator. In randomized order, both investigators stimulated each subject's median nerve 10 times, once every 15 sec. Each session included the measurement of the subject's flexor carpi radialis maximal M-wave amplitude and H-reflex amplitude and latency with surface electromyographic electrodes. The intraclass correlation coefficients (ICC) indicated an adequate correlation between investigators for both M-wave maximal amplitude and H-reflex at 5% of the M-wave maximal amplitude (.84 and .70, respectively). However, there was a low correlation (.38) between the latency values obtained by the two investigators. The peak-to-peak amplitudes of the H-reflex and M-wave do not appear to be influenced by experience of the tester. The latency of the response, however, appears to have an associated learning curve, improving in consistency with increasing practice of tester.

Reliability of the biceps brachii M-wave.

BACKGROUND: The peak-to-peak (P-P) amplitude of the maximum M-wave and the area of the negative phase of the curve are important measures that serve as methodological controls in H-reflex studies, motor unit number estimation (MUNE) procedures, and normalization factors for voluntary electromyographic (EMG) activity. These methodologies assume, with little evidence, that M-wave variability is minimal. This study therefore examined the intraclass reliability of these measures for the biceps brachii. METHODS: Twenty-two healthy adults (4 males and 18 females) participated in 5 separate days of electrical stimulation of the musculocutaneous nerve supplying the biceps brachii muscle. A total of 10 stimulations were recorded on each of the 5 test sessions: a total of fifty trials were used for analysis. A two-factor repeated measures analysis of variance (ANOVA) evaluated the stability of the group means across test sessions. The consistency of scores within individuals was determined by calculating the intraclass correlation coefficient (ICC). The variance ratio (VR) was then used to assess the reproducibility of the shape of the maximum M-wave within individual subjects. RESULTS: The P-P amplitude means ranged from 12.62 +/- 4.33 mV to 13.45 +/- 4.07 mV across test sessions. The group means were highly stable. ICC analysis also revealed that the scores were very consistent (ICC = 0.98). The group means for the area of the negative phase of the maximum M-wave were also stable (117 to 126 mV.ms). The ICC analysis also indicated a high degree of consistency (ICC = 0.96). The VR for the sample was 0.244 +/- 0.169, which suggests that the biceps brachii maximum M-wave shape was in general very reproducible for each subject. CONCLUSION: The results support the use of P-P amplitude of the maximum M-wave as a methodological control in H-reflex studies, and as a normalization factor for voluntary EMG. The area of the negative phase of the maximum M-wave is both stable and consistent, and the shape of the entire waveform is highly reproducible and may be used for MUNE procedures.

Reliability of the FCR H-reflex.

This study examined the intraclass reliability of the latency and amplitude of the Hoffmann reflex (H-reflex) in the flexor carpi radialis (FCR). The stability and consistency of the latency and peak-to-peak amplitude of the H-reflex were assessed across four test sessions. The FCR H-reflex was evoked in 39 volunteers (20 males and 19 females) on four separate days. The maximum amplitude of the motor response (Mmax) was determined each day and 10 responses were recorded at that stimulus intensity. The H-reflex was then evoked at an intensity corresponding to 5% of Mmax (H5%) and 10 recordings were obtained. The latency of the H-reflex and the peak-to-peak amplitude of both Mmax and H5% were calculated for each trial. Determination of reliability involves the consideration of both the stability and consistency of the measures. The reliability of the measures in this investigation was assessed using an analysis of variance and corresponding Lindquist intraclass correlation coefficient (ICC) model. In contrast to previous investigations, the H-reflex was evoked without facilitation in 95% (37/39) of the subjects tested. Statistical evaluation revealed that the latency and amplitude of the H-reflex, as well as the amplitude of Mmax, were both stable and consistent across the four test days. The ICC for Mmax was 0.97. The ICC for H5% was 0.92, and for latency of the H-reflex was 0.89. It was shown that the H-reflex can be easily evoked in the FCR and that the latency and amplitude of these recordings are highly reliable. The demonstration that the H-reflex can be easily and consistently evoked in the FCR has important clinical implications. It provides a tool for clinicians to assess the C7 level of the spinal cord and median nerve function.

Rapid upregulation of pyruvate dehydrogenase kinase activity in human skeletal muscle during prolonged exercise.

Prolonged moderate-intensity exercise is characterized by a progressive reduction in carbohydrate oxidation and concomitant increase in fat oxidation. Pyruvate dehydrogenase (PDH) controls the entry of pyruvate into oxidative pathways and is a rate-limiting enzyme for carbohydrate metabolism. PDH is controlled by the activities of a kinase (PDK, inhibitory) and phosphatase (stimulatory). To test the hypothesis that increased PDK activity was associated with decreased PDH activity and carbohydrate oxidation during an acute exercise bout, seven recreationally active men completed 4 h of cycle exercise at 55% peak oxygen consumption. Muscle samples were obtained before and at 10 min and 4 h of exercise for the measurement of PDH activity and the extraction of intact mitochondria for the measurements of PDK activity and PDK-2 and PDK-4 protein expression. Carbohydrate oxidation was reduced (P < 0.05) with exercise duration. Muscle glycogen content was lower (P < or = 0.05) at 4 h compared with rest and there was no change in muscle pyruvate content from 10 to 240 min during exercise (10 min: 0.28 +/- 0.05; 240 min: 0.35 +/- 0.09 mmol/kg dry muscle). PDH activity increased (P < 0.05) above resting values at 10 min (2.86 +/- 0.26 mmol.min(-1).kg wet muscle(-1)), but was lower than 10 min after 4 h (2.23 +/- 0.24 mmol.min(-1).kg wet muscle(-1)) of exercise. PDK-2 and PDK-4 protein expression was not different from rest at 10 min and 4 h of exercise. PDK activity at rest averaged 0.081 +/- 0.016 min(-1), was similar at 10 min, and increased (P < 0.05) to 0.189 +/- 0.013 min(-1) at 4 h. Although reduced glycolytic flux may have played a role in decreasing carbohydrate oxidation, the results suggest that increased PDK activity contributed to the reduction in PDH activity and carbohydrate oxidation late in prolonged exercise. The increased PDK activity was independent of changes in intra-mitochondrial effectors, and PDK-2 and PDK-4 protein content, suggesting that it was caused by a change in the specific activity of the existing kinases.