The three main metabolic systems providing ATP either at rest or in muscle exercise (phosphocreatine (PCr) hydrolysis, anaerobic glycolysis, and aerobic oxidative metabolism) are recruited differently according to the muscle characteristics, the exercise modality, and the training status (29). It is well established that human skeletal muscle is a heterogeneous tissue and can adapt to the variable functional requirements through mechanisms based on changes in muscle mass, fiber size, fiber type distribution, metabolic enzyme activities, and substrate supply. Human muscles are mixed muscles expressing three main fiber types: type I, IIA, and IIX, in variable proportions (7). They exhibit increasing shortening velocities in this rank order. The relative importance of the two main metabolic pathways of ATP production, that is, anaerobic glycolysis and aerobic oxidative metabolism, varies between muscle types. The slow type I fibers can sustain prolonged low-power work in association with a well-developed oxidative metabolism. The fast type IIX fibers are adapted to brief and intense contractions fueled by the anaerobic glycolytic pathway and immediate availability of PCr; type IIX fibers are much better used for fast and powerful movements (7). Type IIA fibers are fast oxidative-glycolytic and exhibit intermediate contractile function. Because short-duration, maximal-intensity exercise involves the recruitment of all the fiber types (10), the resultant metabolic response needs to be investigated in more detail, particularly in subjects whose responses may be affected by training adaptations.
Currently, the available evidence indicates that metabolic diversity involves many different aspects of muscle fiber metabolism, from substrate availability to the rate of enzymatic process of energy production and use (7). Glycogen content at rest is higher in fast than in slow fibers, and it was demonstrated that endurance training increases glycogen content and the glycolytic enzyme activities (37). Within the skeletal muscle cell at the onset of muscular contraction, PCr represents the most immediate reserve for the rephosphorylation of ATP. Nevertheless, muscle fibers differ in the availability of PCr. Several studies consistently indicate that in human muscles, resting PCr content is greater in fast than in slow fibers (7,26) and that sprint training increases the muscle PCr content (17). On exercise, the PCr content decreases to reach similar values in both fast and slow fibers. In contrast, resting ATP is similar in slow and fast fibers. Glycolytic and oxidative ATP synthesis allows PCr recovery. The factors affecting the rate of PCr resynthesis after intense exercise have recently been reviewed (26). The recovery is faster in slow than in fast fibers in the first minute after maximal exercise. The information currently available for strength exercise in vivo is limited to the postexercise phase.
So far, there is no reliable method for quantifying the instantaneous contribution of different fiber types on muscle recruitment and the resulting overall metabolic responses in vivo. The most useful techniques capable of measuring muscle metabolism noninvasively are 31P magnetic resonance spectroscopy (31P-MRS), 1H magnetic resonance imaging (1H-MRI), proton magnetic resonance spectroscopy (1H-MRS), and near-infrared spectroscopy (NIRS). However, these techniques do not allow direct discrimination of metabolic changes at the level of type I or type II fibers in vivo. The 31P-MRS measures muscular intracellular pH and the concentrations of the main phosphorylated compounds with a poor time resolution (longer than 1-2 s) (13). In addition, the compounds can only be quantified if the specific saturation factors remain constant during the exercise. The 1H-MRI provides anatomic information (and also microvascular density) as well as muscle perfusion/oxygenation. However, being sensitive to motion artifacts, 1H-MRI can be used only to investigate the postexercise recovery with a time resolution of about 0.03 s (13,38). The 1H-MRS measures muscle deoxymyoglobin signal, allowing the assessment of intracellular O2 availability at rest (33) and during exercise with a poor time resolution (longer than 12 s) (9). NIRS measures muscle oxyhemoglobin (O2Hb) saturation (SmO2), concentration changes of O2Hb, deoxyhemoglobin (HHb), and total hemoglobin volume (tHb = O2Hb + HHb), and, indirectly, blood flow and O2 consumption (12,28,31). Therefore, NIRS has largely been used in muscle exercise pathophysiology (5,8,12,30). NIRS is a relatively low-cost technique, and it is less sensitive to motion artifacts than are 31P-MRS/1H-MRI/1H-MRS. Moreover, NIRS can reach a time resolution of about 0.1 s, allowing the measurement of SmO2 kinetics during the natural execution of whole-limb or whole-body exercise (not feasible inside the magnet by 31P-MRS, 1H-MRI, and 1H-MRS), including very short exercises.
It is well established that skeletal muscle can adapt to variable functional requirements, even through changes in fiber-type characteristics and in contractile function. In particular, high-intensity exercise training increases the ability to develop force rapidly (7), the maximal integrated electromyography activity, and the cross-sectional area of the muscle (by 12% after 12 wk) (34). For example, it has recently been reported that the vastus lateralis (VL) muscle of bodybuilders was markedly adapted to hypertrophic heavy resistance exercise through 1) an extreme hypertrophy, 2) a shift towards the stronger and more powerful fiber types (IIX), and 3) an increase in specific force of muscle fibers (11). One-leg, heavy-resistance strength training provoked hypertrophy of VL muscle fibers after 9 wk of training; the cross-section area of type I, IIA, and IIX fibers increased by 18, 21, and 41%, respectively, with no significant changes in fiber-type distribution (25). Resistance training induced an increase in the VL muscle capillarization (16) and leg myoglobin (Mb) concentration (9). So far, the effects of training on muscle metabolism during intense exercise remain controversial (17,21). During cycling, the anaerobic ATP production was found to be reduced in subjects after sprint training (17). In contrast, during maximal isometric contractions, the anaerobic ATP production was found to be two- to threefold higher in sprint-trained subjects compared with endurance-trained or untrained subjects (21).
The metabolic changes induced by heavy-resistance, systematic strength training have mainly been investigated by 31P-MRS using tests of prolonged duration (from about 30 s up to several minutes) (21,36). Towse et al. (38) have found, by 1H-MRI and NIRS in sedentary and active subjects, a similar postexercise muscle O2Hb desaturation after 1 s of ankle dorsiflexion of the anterior tibialis muscle. The postexercise transient hyperemia measured by 1H-MRI was more than threefold greater in active compared with sedentary subjects. To the best of our knowledge, there are no studies on the recruitment of the diverse skeletal muscle metabolic systems during very short, maximal, voluntary contractions.
The main purpose of this study was to investigate the effects of a brief and fast maximal isometric voluntary contraction on VL SmO2 (measured by NIRS) in heavy-resistance strength-trained and untrained subjects. On the basis of the literature, we assumed that the heavy-resistance strength-trained subjects would have, with respect to the untrained subjects, 1) an increased ability to use nonoxidative resources and/or an increased supply of substrate to be used nonoxidatively; 2) a higher PCr concentration on average; 3) a hypertrophy of type I, IIA, and IIX fibers; 4) a higher capillarization; and 5) a higher myoglobin content. Therefore, we hypothesized that we would find a delayed beginning of the use of the aerobic oxidative metabolic system (evaluated as muscle O2Hb desaturation) in the VL muscle of the heavy-resistance strength-trained subjects during a very short maximal contraction that would provoke a complete occlusion of the blood flow.
MATERIALS AND METHODS
Two groups of healthy male volunteers (trained and untrained) took part in this study. The 10 trained subjects were involved in heavy-resistance strength-training programs for more than 1 yr (> 2 h·d−1; 3-7× wk−1). The 10 untrained subjects were university students who did not spend any time on specific physical exercise. The two groups were not statistically different by unpaired t-test for age (P = 0.97), height (P = 0.30), or weight (P = 0.07). Mean (± SD) age, height, and body mass of all the subjects were 28.0 ± 6.3 yr, 1.8 ± 0.1 m, and 77.8 ± 9.9 kg, respectively. After receiving a complete explanation of the purpose and the procedures of the study, subjects gave their written consent; none of them reported recent thigh injuries. The study was approved by the university ethics committee and conformed to the regulations laid out in the Declaration of Helsinki on the use of human subjects in research.
All subjects performed five trials on a modified leg press machine (Leg Press, TechnoGym, Italy). Every trial consisted of 1) a 1-min rest period, 2) a leg press exercise of approximately 2-4 s, and 3) a 2-min recovery period. The leg press exercise consisted of a static maximal voluntary contraction (MVC), for which the subjects were instructed, on verbal command, to act against the footplate "as forcefully and as fast as possible" using only their dominant leg, and to maintain the MVC for a time period of approximately 2-4 s while the other leg was resting. The dominant leg was determined by the Edinburgh dominance test (32), to which specific items were added regarding foot preference. During the trial duration, volunteers were positioned on the seat of the leg press with a knee angle of 110° (full extension being 180°). Knee joint angle was determined with a handheld goniometer. The subject waist was fixed in position by bands; during the exercise, the subjects were allowed to stabilize their upper body by holding the handles of the leg press machine. NIRS recording was interrupted at the end of the 2-min postexercise recovery period of each trial. Subjects then remained seated, moving freely and relaxing their legs, for the next 2 min. The fifth minute of recovery corresponded to the rest period of the subsequent trial. During the intertrial periods, cardiac frequency was monitored by a pulse oximeter (N-200; Nellcor, Pleasanton, CA), with the sensor attached on the forehead. Cardiac frequency returned back to baseline values at the end of the rest period (data not shown). Neuromuscular and metabolic fatigue was considered to be minimal because of 1) the small number of repetitions performed, 2) the sufficient rest interval between trials, and 3) the good health of all the subjects. The sufficient rest interval between trials was confirmed by post hoc analysis, which demonstrated that the mean tissue oxygenation index (TOI) values, evaluated from −4 to −1 s before each MVC, returned to the baseline values (data not shown). Subjects were familiarized with the protocol before the beginning of the study. The first trial was preceded by a 10-min warm-up performed on an electronically braked cycle ergometer (Ergocard II, OTE Biomedica, Italy; 50 W, 50-60 rpm).
Leg press strength was recorded using a calibrated load cell (UU-K300, Dacell, Korea) inserted into the mechanical vinculum that fixed the seat to the foot board of the leg press machine. The force signal was monitored and recorded (at a rate of 100 Hz) by means of a digital oscilloscope (TDS 210, Tektronix Inc, Beaverton, OR); the data were stored on a personal computer for offline analysis. Force transducer calibration was performed according to the manufacturers' specifications. In particular, the electrical signal, produced by the transducer when 100-, 500-, 900-, 1500-, 2100-, and 3300-N loads were placed on the load cell, was recorded to determine the relationship between the force applied and the transducer's output. Before every testing session, the reproducibility of the measure was verified by applying a 1500-N load. The measured linearity and repeatability were better than 0.3% and 0.2%, respectively. For each trial, the force was continuously measured at least 5 s before, during, and 5 s after the exercise.
The NIRS measurements were performed with a NIRO-300 oximeter (Hamamatsu Photonics K.K., Japan) (35). After the probe testing on a phantom to analyze the total probe sensitivity and the sensitivity difference between the three sensors of the detection probe, the optical probe (consisting of one emitter and one detector, 4.5 cm apart), supported by a rigid rubber shell, was firmly attached to the skin overlying the belly of the VL muscle, parallel to the major axis of the thigh and at 14-20 cm from the knee, by a double-sided adhesive sheet. The rigid rubber shell, in turn, was secured by a soft and elastic bandage (Tensoplast, BSN Medical, South Africa). For identification of the VL muscle belly, on the day before the measurements, each subject was asked to perform a preliminary leg press isometric contraction. The identified area was carefully shaven before the experimentation. A pen mark was used to indicate the correct position of the probe holder shape for the next day. Pen marks were also made on the skin to check for any sliding of the rubber shell during the exercise. No sliding of the probe was observed at the end of the measurements in any subject. The NIRS data were recorded with a sampling rate of 6 Hz. Events were marked on the NIRO-300 to indicate the start/stop of each trial and the onset/end of each exercise. The measured NIRS data by the NIRO-300 were 1) SmO2 as TOI (expressed as a percentage), 2) concentration changes in O2Hb and HHb (expressed in micromoles per liter per centimeter), and 3) the derived changes in tHb (expressed in micromoles per liter per centimeter). These measurements can be affected by the contribution of Mb oxygenation changes (9,12). TOI reflects the local balance between O2 supply and O2 consumption. The tHb volume changes, being strictly related to blood volume changes, can be considered an indirect measure of changes in local blood flow. After fixing the probe holders on the subjects, an initialization procedure was carried out. The latter sets each laser power automatically, establishing the optimum measurement condition. The zero-set procedure (carried out just before the beginning of each baseline condition) was adopted to return the O2Hb, HHb, and tHb parameters to the zero value. This procedure does not affect the TOI value, because TOI is measured as an absolute value instead of a change with respect to the arbitrary initial zero value. The adipose tissue thickness overlaying the VL muscle was measured with a skinfold caliper (British Indicators Harpenden, UK). An unpaired t-test established that there was no difference between the two groups (P = 0.11). The adipose tissue thickness was 3.1 ± 0.8 mm (mean ± SD). Considering that the adipose tissue thickness was relatively low, and the penetration depth of the NIRS signal is almost half of the source-detector separation (4.5 cm in the optical probe of the NIRO-300), the changes in TOI and tHb reflected mainly muscle metabolic and hemodynamic changes of VL (12).
Strength and NIRS data were exported in text format without filtration. Analysis was performed with custom software developed in Matlab (version 5.3, The MathWorks Inc.).
Force data analysis.
Each force dataset was expressed in newtons (by the measured calibration factors) and smoothed (Savitzky-Golay filter, polynomial order 2, frame size 41). The resting mean and SD force values were determined in the time interval from 1 to 4 s after the start of the recording of the force signals (Figs. 1 and 2A). This interval terminated around 1 s before the MVC onset. The MVC onset was identified as the force value higher than the threshold and maintained for at least 1 s. The threshold was considered the rest mean force value plus two times the rest SD value. The rest 2-SD value range was 170-269 and 100-243 N for trained and untrained subjects, respectively. The mean and SD of the force expressed during each exercise were calculated for 1.5 s of the exercise in the time interval from 0.5 to 2.0 s after the MVC onset. The exercise end was identified as the time when the force value became lower than the mean, minus 2 SD of the force expressed during the exercise. For each trial, the following force parameters were considered: 1) mean force output (Fm), 2) force duration (TF), and 3) maximum rate of force development (RFD). Fm was the mean force expressed during the specified 1.5 s of exercise minus the rest mean force. TF was the difference between the end and the onset of exercise according to the force data. RFD was the first derivative maximum of the force-time dataset. For each subject, the reported force and NIRS data are referred to the best performance trial with regard to the maximal RFD value.
NIRS data analysis.
The mean and SD values of resting tHb and TOI were calculated for the time interval from −4 to −1 s (Figs. 1 and 2B and C) before the exercise-onset event marker. This rest interval terminated around 1 s before the MVC onset. The onset of the exercise was identified as the time at which the tHb values became, for at least 1 s, lower than the mean minus two times the SD of the resting tHb. The rest 2-SD value range was 9-46 and 8-37 μM·cm for trained and untrained subjects, respectively. The mean and SD values of tHb and TOI occurring during each MVC were calculated for the time interval from 0.5 to 2.0 s after the identified onset of the exercise. The exercise end was identified as the time at which the tHb values became, for at least 1 s, higher than mean plus two times the SD value of the tHb expressed during the exercise. The onset of TOI decrease was identified as the time at which the TOI values became, for at least 1 s, lower than the mean minus two times the SD of the resting TOI. The rest 2-SD value range was 1.2-2.4% and 1.9-2.5% for trained and untrained subjects, respectively. For each analyzed trial, the following NIRS parameters were considered: 1) mean tHb and TOI changes during exercise (ΔtHbmean and ΔTOImean, respectively), 2) contraction duration based on NIRS data (TtHb), 3) time to the onset of TOI decrease (TTOI), 4) maximum postexercise TOI decrease (ΔTOImin), 5) time to maximum postexercise TOI decrease (TTOImin), 6) half-recovery time of TOI (T1/2TOI), 7) maximum postexercise tHb increase (ΔtHbmax), and 8) time to maximum postexercise tHb increase (TtHbmax). ΔtHbmean and ΔTOImean were the tHb and the TOI values, respectively, calculated as means for the specified 1.5 s of exercise minus the corresponding mean rest values. TtHb was the difference between the end and the onset of the exercise based on tHb signal. TTOI was the onset of TOI decrease minus the onset of the exercise for NIRS data. The ΔTOImin was the difference between the minimum TOI value reached during the postexercise phase and the corresponding mean resting value. TTOImin was the time at which TOI reached its minimum value during the postexercise phase. The T1/2TOI was the time at which TOI recovered 50% of ΔTOImin from minimum TOI. The ΔtHbmax was the difference between the maximum tHb value reached during the recovery phase and the corresponding mean resting value. TtHbmax was the time at which tHb reached its maximum value during the recovery. TTOImin, T1/2TOI, and TtHbmax were counted from the onset of the exercise as identified by using the tHb values.
Statistical analyses were performed using the SigmaStat 3.5 package (Systat Software Inc., Richmond, CA). The average values were expressed as means ± SD. A one-way analysis of variance (ANOVA) was used to compare the data between the two groups of subjects (training effect). A two-way ANOVA was used to compare the data between groups and the order trial selected (trial effect). A two-way repeated-measures ANOVA was used for comparisons within subjects (signal or exercise effect) and groups, followed by the Student's t-test when appropriate. Results were considered statistically different at P < 0.05. Bland and Altman's limits-of-agreement plot (6) was employed to assess the level of agreement between the exercise duration determined using the force-time curve and the tHb-time curve.
Figures 1 and 2 show a typical time course of leg force and TOI, as well as O2Hb, and HHb changes (used to calculate tHb changes) measured on a trained subject (Fig. 1) and an untrained subject (Fig. 2). These subjects were chosen as representative of the difference between the two groups. The diverse force performance between the trained and untrained subjects is shown by the dissimilar RFD values (about 7800 and 5000 N·s−1 in the trained and untrained subject, respectively). The onset of the exercise clearly provoked an abrupt drop of tHb, which could be attributable mainly to venous compression. tHb was almost stable for the exercise duration and promptly started to increase after the exercise end; then, a transient tHb overshoot was observed. tHb returned slowly to its preexercise level within 1-3 min (data not shown). On the other hand, in the trained subject TOI was almost stable during the exercise period (force duration, 3.13 s) and started to decrease 3.96 s after the beginning of the exercise, whereas in the untrained subject (force duration, 3.21 s) TOI started to decrease at 2.48 s. After the end of the short exercise bout, in both subjects TOI consistently and progressively decreased, reaching actual minimum values of 63.4 and 51.6% in 11.39 and 9.74 s in the trained and untrained subject, respectively; then, TOI progressively returned to the preexercise level in about 1-1.5 min.
Table 1 reports the leg force and VL muscle oxygenation/hemodynamic parameters for the best performed trial with regard to the highest RFD value. No relationship between the two groups was found regarding the chronological order of the trial associated with the highest RFD value (P = 0.73). The analysis of variance revealed a significant difference in RFD between the exercises performed by the trained and untrained subjects (P < 0.05). In contrast, Fm and TF were not significantly different between the two groups (P = 0.28 and 0.14 for Fm and TF, respectively). The duration of the exercise, according to tHb changes, was not significantly different between the two groups (P = 0.25). Moreover, TtHb was significantly higher than TF within each group (P < 0.001). However, the Bland-Altman plot between TtHb and TF (Fig. 3) shows 1) no trend in the data as the mean time duration increases, 2) a 95% confidence interval from −0.14 to + 0.42 s, and 3) a bias of +0.14 s. The latter can be explained by the different sampling times of the NIRO-300 and the force transducer (0.16 s and 0.01 s, respectively).
At rest condition, a significant interindividual variation in the TOI percentage was found in both groups (73.5 ± 5.1 and 71.4 ± 4.4% in the trained and untrained groups, respectively). The maximum TOI achieved during exercise with respect to the rest condition was not different between the two groups (2.5 ± 1.3 and 2.8 ± 1.3% in the trained and untrained groups, respectively; P = 0.60). During the exercise, tHb and TOI values changed significantly with respect to the rest condition (P < 0.001 and P < 0.01 for tHb and TOI, respectively). The amplitude of these changes was similar in both groups (P = 0.95 and 0.35 for tHb and TOI, respectively). The time to the onset of TOI decrease was consistently shorter in the untrained subjects than in the trained ones (Table 1; P < 0.01). This difference is better illustrated by Figure 4, which shows the time to the onset of the TOI decrease versus the exercise duration measured in all the subjects. Nine measurements collected on trained subjects are above the identity line (with two data points overlapping), and one is coincident with the identity line. Six measurements collected on untrained subjects are below the identity line, two are coincident with the identity line, and two are above, though very close, to the identity line.
Maximum postexercise TOI decrease was consistently greater in the untrained subjects, and the time to maximum postexercise TOI decrease was consistently longer in the trained subjects (Table 1; P < 0.05 and P < 0.01 for ΔTOImin and TTOImin, respectively). Half-recovery time of TOI was similar in both groups (P = 0.06). Figure 5 reports the maximum postexercise TOI decrease (ΔTOImin, %) versus half-recovery time of TOI (T1/2TOI, s) for trained and untrained subjects. Maximum postexercise tHb increase and time to maximum postexercise tHb increase were not significantly different between trained and untrained subjects (P = 0.96 and 0.37 for ΔtHbmax and TtHbmax, respectively).
These results are consistent with our hypothesis that, on a brief and fast maximal isometric voluntary contraction, VL muscle of heavy-resistance strength-trained subjects would have, with respect to the VL muscle of untrained subjects, a delayed beginning of the use of the aerobic oxidative metabolic system (evaluated as muscle O2Hb desaturation). In particular, the SmO2 of the VL muscle of the trained group started to decrease only after the end of the exercise in 9 of 10 subjects, and just at the end of the MVC in the remaining subject (Fig. 4). In the case of the untrained group, SmO2 started to decrease before the end of the exercise in 8 of 10 subjects only. To the best of our knowledge, this is the first time that the characteristics of NIRS of high temporal resolution and low sensitivity to motion artifacts have been exploited to investigate the time course of SmO2 during an explosive intense exercise.
The maximum rate of force development is an important strength parameter because it incorporates the aspect of contraction time, which is neglected using the Fm (1). RFD describes the ability to rapidly develop muscular force and reflects the ability of a subject to realize the contraction "as forcefully and as fast as possible." So, the maximum RFD value can be considered one of the most reliable muscle force-dependent tests of explosive force production (27). According to previous studies (2,27), in the present study all five trials were used for the assessment of the RFD reliability, whereas the trial associated with the highest RFD value served for the data analysis. The relatively large SD of the RFD values found in the present study (Table 1) is in agreement with values reported in recent similar studies (2,27,38). Although the rapid exertion of maximum force requires more practice than exerting force per se, this intersubject variability of RFD values could be explained by the diversity of one or more of the following physiological factors: muscle fiber type and myosin heavy-chain composition, muscle cross-sectional area, viscoelastic properties of the muscle-tendon complex, and neural drive to the muscle (2). Strength and endurance training produce widely diversified adaptations, with little overlap between them (29). Strength training typically results in increases of muscle mass and muscle strength. In contrast, endurance training induces increases in maximal O2 uptake and metabolic adaptations that lead to an increased exercise capacity. Training induces gains in RFD (1,34), so, as would be expected, in the present study the heavy-resistance strength-trained subjects developed an RFD higher than that produced by the untrained subjects.
The mean force output was not significantly greater in the trained versus untrained subjects (Table 1) and was highly correlated with the maximum force achieved during MVC (P < 0.001, R = 0.97; data not shown). Similar results were found by others when the force output was collected from different groups of subjects (active vs sedentary) (38). Therefore, because the Fm of the two groups was similar (at least for the time period between 0.5 and 2 s of MVC), and assuming that the exercise energetic cost was not substantially different between the two groups (21), the delayed time to onset of TOI decrease in the trained group suggests that the VL muscle of the trained subjects, during a very short and fast high-intensity isometric contraction, uses other metabolic systems rather than the aerobic oxidative one. The delayed activation of the aerobic oxidative metabolic system (evaluated as muscle O2Hb desaturation) in the trained subjects could be a consequence of a larger proportion of type II fibers and a higher PCr concentration on the average, or greater PCr breakdown and anaerobic glycolytic ATP provision in trained than in untrained subjects.
Our results are also in agreement with a recent 31P-NMR study (21) that demonstrates marked differences in force production, aerobic as well as anaerobic gastrocnemious muscle metabolism, between endurance-trained, sprint-trained, and untrained subjects during four maximal isometric contractions of 30-s duration each. No difference was found in resting PCr/ATP among the three groups. The groups differed with respect to PCr breakdown; sprinters demonstrated about 75% breakdown in each contraction compared with about 60 and 40% for untrained and endurance-trained subjects, respectively. In particular, during the first 5 s of MVC, PCr decreased by about 10% and pH increased by about 0.1 units in sprint-trained subjects. Conversely, PCr and pH were unchanged during MVC in the untrained subjects, suggesting that the aerobic system started to be used.
A delayed VL muscle desaturation has also been observed at the onset of cycling exercise by Grassi et al. (14). The authors consider PCr hydrolysis and anaerobic glycolysis responsible for the delay or the attenuation of the increase in ADP concentration within the cell after rapid increase in ATP demand, thereby "buffering" a more rapid activation of oxidative phosphorylation. In isolated single-frog myocytes, it was found that at the contraction onset, a monoexponential decrease in intracellular PO2 was preceded by about 10 s in which PO2 remained constant (23,39). The results of those studies suggest that there was plenty of O2 available at the mitochondrial level in these cells during the first seconds of contraction (39). Therefore, in the subjects of the present study, the exploitation of the aerobic oxidative metabolism, which uses O2 available in the mitochondrial matrix, could not be excluded during the very short exercise. If stored oxygen is greater in the trained subjects, this may partly explain a later onset of TOI decrease.
In human skeletal muscle, the role of Mb and its relationship with factors such as muscle perfusion and metabolic capacity are not yet well understood (40). Unfortunately, NIRS is unable to differentiate between the signal attenuation attributable to Hb and Mb, because the absorbency signals of these two chromophores overlap in the near-infrared range. Mb is a confounding factor at 10% of the whole NIRS signal (12). Considering the poor temporal resolution of 1H-MRS, no data are available on Mb desaturation during very short MVC (9). The present NIRS results collected on trained subjects suggest that although VL muscle blood flow was interrupted, Hb and Mb did not desaturate during the 2-4 s of high-intensity exercise. During the recovery of high-energy phosphate levels after exercise, Mb is expected to be fully saturated and does not affect the whole NIRS signal. Recently, leg Mb concentrations, measured by NMR, were shown to be 25% greater in endurance-trained athletes as compared with sprinters, and to correlate with mitochondrial oxidative production of ATP (9). We could speculate that in our study the heavy-resistance strength-trained subjects might also have higher Mb concentration compared with untrained subjects. Combined 1H-MRS and NIRS studies are needed to clarify not only the issue of the contribution of Mb to the NIRS signal, but also the kinetics and the amount of Mb desaturation during exercises with different workloads (9).
An accurate determination of the beginning/end of the MVC as well as the stability of the blood volume (measured as tHb changes) during the exercise are both necessary to claim the validity of our results. It is well known that the muscle contraction pinches the arteries/arterioles and veins/venules along the fascicular lines, whereas the capillaries are relatively uncompressed (15). A VL muscle maximal isometric voluntary contraction provokes a complete obstruction of muscle blood flow (3,4). This is witnessed by the fact that tHb, after an immediate initial drop, was stable during the exercise period. The exercise duration was determined either on force or tHb time courses (Figs. 1 and 2). The beginning of the exercise evaluated on the basis of both force and tHb tracings was coincident for the immediate effect of the muscle contraction pinch on the muscle blood volume. Although TtHb was higher than TF, the Bland-Altman plot shows a small bias attributable to the different sampling times of the NIRS instrumentation and the force transducer (Fig. 3).
A diverse postexercise TOI decrease (less intense in the trained subjects) was found (Table 1 and Fig. 5). Figure 5 clearly illustrates the large data-point dispersion in ΔTOImin, which is also confirmed by the high SD reported in Table 1. The half-recovery time of TOI was not different between trained and untrained subjects. The presence of a data point with a prolonged recovery time (about 50 s) might be explained by the subject's difficulty to relax the VL muscle immediately after the exercise. A similar SmO2 time course was also found in the finger flexor muscle after a 10-s MVC (22). In that study, a significant positive correlation between postexercise SmO2 recovery rate (measured by NIRS) and the time constant for PCr resynthesis (measured by 31P-MRS) was found. The postexercise SmO2 recovery rate closely correlated with muscle O2 consumption, but not with forearm blood flow. A delayed SmO2 recovery has recently been reported in the tibialis anterior muscle after a 3-s MVC (24).
Glycolytic and oxidative ATP synthesis allows PCr recovery after MVC. The recovery is faster in slow than in fast fibers for the first minute after maximal exercise (7). The factors affecting the rate of PCr resynthesis after intense exercise have been recently reviewed (26). In our study, the O2 supply is limited during the early phase of post-MVC metabolic recovery in the VL muscle of trained and untrained subjects, because TOI transiently decreased after the MVC. On the other hand, it has been reported that PCr recovery is limited by O2 availability in trained subjects (18) and by mitochondrial oxidative capacity in untrained subjects (19). In fact, it has been demonstrated in exercise-trained subjects that PCr recovery occurred more rapidly when inspired oxygen fraction was 1.0 than when it was 0.21 (18).
The time course of the TOI recovery depends also on the post-MVC muscle blood-flow increase. It is generally accepted that tHb changes are related to blood-flow changes (12). Taking into account that no differences were found in the increase of the maximum postexercise tHb and its corresponding time between trained and untrained subjects (Table 1), the differences in postexercise TOI decrease cannot be attributable to differences in postexercise muscle blood flow. The differences in the VL muscle oxygenation recovery after maximal cycling exercise found between sedentary and active subjects (20) could be explained by differences in postexercise muscle blood flow on prolonged exercise and/or muscle capillarization. In our study, the very short duration of the VL muscle isometric exercise (3 s vs several minutes) was not capable of generating significant postexercise muscle blood-flow differences, as indirectly indicated by the tHb changes. Conversely, Towse et al. (38) have recently found, by 1H-MRI, a postexercise transient hyperemia more than threefold greater in active compared with sedentary subjects after a 1-s ankle dorsiflexion of the anterior tibialis muscle. Nevertheless, any assumptions done concerning the training-induced adaptations that would have influenced the results between the two groups remain to be verified in future studies by invasive or other measurements.
The major advantage of NIRS is represented by the possibility to perform tissue oxygenation measurement repeatedly. The main NIRS results in sports science have been reported and discussed in several recent reviews (5,30). Despite the advantages of NIRS, there are limitations of this study that warrant discussion. Some of the limitations are related to the general assumptions made in NIRS. 1) Absorbing compounds: Hemoglobin is considered the main absorbing component in the tissue volume interrogated. 2) Sample volume: The use of NIRS allows the investigation of only a few cubic centimeters of superficial VL muscle. Therefore, it is assumed that the investigated portion of a given muscle is recruited in proportion to the work performed. 3) The accuracy of TOI measurement relies on the assumption that muscle tissue is macroscopically homogeneous: This is not certainly true for in vivo measurement, because of the skin-fat layer separating the NIRS probe from the muscle. However, the multidistance method strongly reduces the effect of the superficial layer on the determination of TOI (35). 4) The difference in the time intervals of the force and the NIRS recordings: In this study, the force and the NIRS data were recorded at 100 and 6 Hz, respectively. Although other NIRS instruments have a higher time resolution (up to 50 Hz) than that of the NIRO-300, they measure concentration changes in O2Hb and HHb only (12). 5) The range in contraction duration between subjects and between trials: It was quite difficult to standardize the duration of a very short MVC. Future studies should be designed to include a variable duration of MVC and intertrial interval. 6) The lack of muscle biopsies or alternative, noninvasive methods to substantiate either metabolic claims or inferences regarding fiber type and recruitment: In the present study, it was not possible to take muscle biopsy samples. 7) Other methods for measuring muscle activity such as EMG would have been beneficial. However, the simultaneous measurement of EMG and NIRS would have been very difficult in the measured muscle because of the size of the optical probe, and it would have required extra trials to be performed by the subjects, to collect EMG information separately. Given the above-mentioned considerations, we are careful to not extend our finding beyond the limitations of the NIRS technology that has been used.
In conclusion, the data from this study suggest that the VL muscle of heavy-resistance strength-trained subjects could have a late activation of the oxidative metabolic system, or greater availability of stored oxygen, during a very fast and short isometric maximal contraction. Considering that leg press exercise 1) is a common exercise used by athletes to enhance performance in sport, 2) is responsible for development of the largest and most powerful muscles of the body, and 3) mimics, from the neuromuscular point of view, many athletic movements, the results of this study could give a valuable contribution to exercise science. For instance, NIRS and leg press exercise could be used to identify, noninvasively, in single muscle groups, the contribution of the aerobic energy system during very short and fast intense exercise, and, secondly, to observe the effects of specific aerobic or anaerobic training programs on the starting time of the aerobic energy system activation.
This research was supported in part by PRIN 2005 and Hamamatsu Photonics K.K. (Japan). The authors wish to thank the two anonymous reviewers for their stimulating comments. The authors also thank Dr. S. Crisostomi and Dr. L. Fallavollita, who assisted the NIRS measurements, and TechnoGym (Italy) for the loan of the leg press machine.
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