Changes in blood volume and oxygenation level in a working muscle during a crank cycle


Medicine & Science in Sports & Exercise:
APPLIED SCIENCES: Physical Fitness and Performance

TAKAISHI, T., T. SUGIURA, K. KATAYAMA, Y. SATO, N. SHIMA, T. YAMAMOTO, and T. MORITANI. Changes in blood volume and oxygenation level in a working muscle during a crank cycle. Med. Sci. Sports Exerc., Vol. 33, No. 3, pp. 520–528, 2002.

Purpose: This study examined circulatory and metabolic changes in a working muscle during a crank cycle in a pedaling exercise with near-infrared spectroscopy (NIRS).

Methods: NIRS measurements sampled under stable metabolic and cadence conditions during incremental pedaling exercise were reordered according to the crank angles whose signals were obtained in eight male subjects.

Results: The reordered changes in muscle blood volume during a crank cycle demonstrated a pattern change that corresponded to changes in pedal force and electrical muscle activity for pedal thrust. The top and bottom peaks for muscle blood volume change at work intensities of 180 W and 220 W always preceded (88 ± 32 and 92 ± 23 ms, respectively) those for muscle oxygenation changes. Significant differences in the level of NIRS parameters (muscle blood volume and oxygenation level) among work intensities were noted with a common shape in curve changes related to pedal force. In addition, a temporary increase in muscle blood volume following a pedal thrust was detected at work intensities higher than moderate. This temporary increase in muscle blood volume might reflect muscle blood flow restriction caused by pedal thrusts.

Conclusion: The results suggest that circulatory and metabolic conditions of a working muscle can be easily affected during pedaling exercise by work intensity. The present method, reordering of NIRS parameters against crank angle, serves as a useful measure in providing additional findings of circulatory dynamics and metabolic changes in a working muscle during pedaling exercise.

Author Information

Institute of Natural Sciences, Nagoya City University, Nagoya 467-8501, JAPAN; Faculty of Economics, Nagoya City University, Nagoya 467-8501, JAPAN; Research Center of Health, Physical Fitness and Sports, Nagoya University, Nagoya 464-8601, JAPAN; Laboratory for Exercise Physiology and Biomechanics, School of Physical Education, Chukyo University, Toyota 470-0348, JAPAN; and Laboratory of Applied Physiology, Graduate School of Human and Environmental Studies, Kyoto University, Kyoto 606-8501, JAPAN

Submitted for publication November 2000.

Accepted for publication July 2001.

Article Outline

Adequate blood supply to working muscles during endurance exercises is essential to support the metabolic requirements and to avoid the decrease in performance caused by muscle fatigue. Previous studies (6,14) have demonstrated that blood flow restriction to working muscles, which is closely related to the development of muscle fatigue, occurs at even low muscle contraction levels (15–22% of maximal voluntary contraction (MVC)). Löllgen et al. (19) have reported a hyperbolic-like relationship between muscular contraction level (peak pedal force) and pedal cadence when reaching a constant work output, and demonstrated that peak pedal forces at 70–100% V̇O2max are produced by only 10–25% MVC under the cadence conditions of 40–100 rpm during pedaling exercise.

In our previous studies, we have demonstrated that the extent of neuromuscular fatigue during pedaling exercise varies among different cadences and minimal fatigue is obtained at a considerably higher cadence (29,30). According to past investigations probing into optimal and/or most economical cadence for (competitive) cycling, it appears that using a higher cadence has some actual advantages in endurance pedaling exercises, such as reducing pedal force (19,28), and increasing the contribution of ST muscle fibers (1) with higher oxidative capacity (17) and mechanical efficiency (9). Additionally, it has been suggested that using a higher cadence contributes to a decrease in intramuscular pressure during muscle contraction and a shortening time for blood flow impairment (14). Considering the outcome of the above research, investigating circulatory dynamics in working muscles in a duty cycle (crank cycle) may provide additional important information in understanding muscle metabolism and alleviating muscular fatigue during pedaling exercise.

Recent physiological studies using a thermodilution technique (3) in circulatory dynamics in working muscles during dynamic muscle contractions (3,18,24) were aimed at investigating the limiting factors of maximal working capacity and evaluating total blood flow returned from the femoral muscles through the femoral vein. To our knowledge, no study thus far has investigated the changes in blood flow in a given muscle during a pedaling exercise against crank angle.

The technological advancement and methodological validity of near-infrared spectroscopy (NIRS) (10,21) enables researchers to estimate relative changes in muscle oxygenation and blood volume during exercise in a noninvasive manner. An example of this methodology has already surfaced in the field of clinical study (32) and exercise physiology (4,5). Recently, Foster et al. (12) described the NIRS technology and claimed that it may have an important role in understanding real-time changes in intramuscular conditions such as intramuscular blood flow or muscle oxygenation while performing competitive efforts. In the NIRS technique, an average value of raw NIRS signal of 10–30 s is generally used as a measurement value. An example of a typical NIRS signal during an incremental pedaling exercise, which resembles the “electromyogram,” was demonstrated by Bhambhani et al. (5) (Fig. 1). Although the change looks noisy, it is expected that the change in NIRS signal will correctly reflect changes in muscle oxygenation at each crank position (crank angle) during a crank cycle.

In the present study, we hypothesized that the “reordered changes in the NIRS parameters” can provide useful and important data for circulatory dynamics and metabolic changes in a given working muscle. The reordered changes in the NIRS parameters are operationalized as a series of NIRS changes reordered at any given crank angle at which time each measurement is taken.

The purpose of this study was to examine circulatory and metabolic changes during a crank cycle in a working muscle by using the reordered changes in NIRS parameters. If the reordered NIRS parameters demonstrate accurate change of biomechanical measurements and neuromuscular activity in the crank cycle, then NIRS can readily be used in circulatory research in sports and exercise science.

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Eight active healthy males (height, 174.3 ± 2.9 cm; body mass, 65.8 ± 3.4 kg; and age, 28.1 ± 5.7 yr (mean ± SD)) with no competitive cycling experience participated in this study. None of the subjects had a history of suffering from injuries during the previous 2 yr. The study was approved by the Institutional Review Board for Use of Human Subjects at Nagoya City University. In addition, all experimental procedures were explained in detail to each subject before signing an informed consent in accordance with the policy statement of the American College of Sports Medicine.

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Experimental Protocols
Maximal exercise test for aerobic power: Max-test.

Each subject performed a preliminary incremental exercise test on a bicycle ergometer (model 818E, Monark, Stockholm, Sweden) at 60 rpm to measure maximal aerobic power (Max-test). After 1 min of unloaded pedaling, work rate was increased to 90 W and afterward gradually increased by 30 W every 2 min until subjects were exhausted, which was defined as the subjects failing to keep a pedal cadence of 60 rpm. Expired gas was sampled by the Douglas bag technique at each 30 s during the exercises when a heart rate (HR) over 175 bpm of was reached. Gas volume was obtained with a dry-method gas meter, and expired O2 and CO2 fractions were measured by a gas analyzer (METs-900, Vise-Medical, Chiba, Japan). Before each Max-test, the gas analyzer was calibrated using two gases of known concentrations. The maximal oxygen consumption (V̇O2max) was confirmed by three criteria: 1) a leveling off or a decrease in V̇O2 despite an increase in work rate; 2) a respiratory exchange ratio (RER) greater than 1.10; and c) attainment of HR higher than 95% of the age-related maximum (220 − age).

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Incremental exercise test for NIRS: MAIN-trial.

On a separate day, subjects performed an incremental exercise test to determine how optical density (OD) data measured by the NIRS device varied with an increasing work rate (MAIN-trial). Following 2 min of rest and 3 min of pedaling at 60 W, work rate was increased by 40 W every 3 min until exhaustion. Subjects were instructed to maintain the pedal cadence of 50 rpm under the auditory guidance of an electric metronome.

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Additional incremental exercise test: Add-trial.

In order to further understand OD changes during a crank cycle, six of the eight subjects participated in an additional incremental exercise test. A duplicate pedaling bout of one work rate (180 W or 210 W) was performed, depending on the subject’s fitness level. Four subjects (subjects A–D) performed a continuous eight bouts of 3 min pedaling at 60, 90, 120, 150, 180, 210, 240, and 180 W, respectively. Two subjects (subjects E and F) performed a continuous nine bouts of 3 min of pedaling at 60, 90, 120, 150, 180, 210, 240, 270, and 210 W, respectively, and cadence was maintained at 50 rpm under the auditory guidance of an electric metronome.

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Cycle ergometer.

A standard friction-loaded cycle ergometer (Monark 818E) was used for the experiments. Four small switches with analog DC output were placed clockwise at 0 (= 360), 90, 180, and 270 degrees from the top position of a crank cycle on the right side of the ergometer to record an accurate time when each crank passed each position during a crank cycle. Analog output from these four switches was continuously digitized by a desktop computer, and the pedal cadence was calculated and numerically monitored on a PC monitor every 6 s for visual feedback.

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Near-infrared spectroscopy.

The NIRS apparatus (HEO-100, Omron, Kyoto, Japan) in the present study consisted of a probe and a computerized control system. The basic principle of this NIRS device has been previously explained by Chance et al. (7) and the device itself described in detail by Hamaoka et al. (15). Briefly, the optical probe consists of a light source and an optical detector both placed to the surface of the tissue. The light source is composed of a pair of two-wavelength LEDs (760 and 840 nm, respectively). The distance between the light source and detector probe is 3.0 cm. The apparatus records an optical density reading during 58 ms at each sampling interval and estimates the changes in hemoglobin (Hb)/myoglobin, oxygenation level (760–840 nm), and muscle blood volume (760 + 840 nm) in tissue.

The reliability of the method of subtracting signals just described has been previously confirmed in in vivo (7) and in vitro experiments (27). The changes in NIRS values were measured during the MAIN-trial and Add-trial by placing the probe over the vastus lateralis muscle 90–120 mm from the knee of the right leg parallel to the major axis of the thigh. The vastus lateralis muscle is generally used for NIRS studies (4,5,7,8) during a pedaling exercise because this muscle is the main contributor of pedal force over the crank angles for pedal thrust (11) among quadriceps muscles. The probe was held with adhesive tape to avoid movement during the trials and covered with a piece of black cloth. All OD data sampled at 2 Hz were stored in a personal computer (PC9801, NEC, Tokyo, Japan), and NIRS values for oxygenation level and muscle blood volume were obtained by a given algorithm (27). The NIRS data sampled at the latter half of each work rate for the MAIN-trial (180 points in total) were analyzed and plotted.

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Analog signal for OD data.

The present NIRS apparatus graphically presented the changes in the NIRS values via the online computer monitor; however, it does not have an electric analog output detecting real-time changes in the OD data. To circumvent this, the beeping signal originating from the NIRS apparatus, which synchronizes the start of each OD data sampling (NIRS sampling time), was used to detect when OD data sampling was performed. After synchronizing the beep sound from an electric metronome to the beep signal (sound) from the NIRS apparatus, the analog signal from the metronome earphone outlet (adjusted to 120 counts·min−1, see Fig. 2) was simultaneously recorded with other analog signals to determine when each OD data was sampled.

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Measurement of EMG.

Myoelectric activity (EMG) during the MAIN trial was recorded by surface EMG technique. The EMG instruments in the present experiment have been fully described in previous studies by the present authors (28). Briefly, two miniature electrodes (Ag-AgCl, 6-mm contact diameter, 3-cm interelectrode distance) were placed over the vastus lateralis muscle just beside the NIRS probe of the right leg, and a reference electrode was place over the anterior superior spine of the iliac crest. All electrode placements preceded a skin abrasion in order to reduce the source impedance to less than 2 kΩ. Myoelectric signals were amplified (AM-601G, Nihon Kohden, Tokyo, Japan) with band pass filtering (5–500 Hz). It was confirmed through various leg muscle contractions that cross-talk of EMGs from other thigh muscles was minimal in each subject and the recorded EMG signals dominantly came from the vastus lateralis muscle. EMG signals of continuous 20 pedal thrusts present at the end of the second minute (90–120 s) and third minute (150–180 s) were used to measure muscular activity and examine neuromuscular fatigue at each work rate for the MAIN-trial. Integrated EMG (iEMG) for each thrust was calculated from the EMG signals between −150 ms and +450 ms of the top-dead center. The time range approximately corresponded to the crank angle from 330 to 150 degrees (Figs. 2 and 3).

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Measurement of pedal force.

Actual force output during pedaling was obtained for each subject through a modified pedal with a standard toe clip mounted on the right crank for the MAIN-trial. Three miniature force transducers (LM-50KA, Kyowa Dengyo, Tokyo, Japan) from inside the pedal and a DC amplifier (DPM-601A, Kyowa) produced an analog signal that indicated the magnitude of the force perpendicular to the pedal (pedal force). Pedal force changes during the stable 30-s period of crank cycles at each work rate were averaged to a single pedal force curve for each subject. Figure 4 shows all eight subjects averaged.

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Measurement of heart rate.

During exercise tests, heart rate was monitored and recorded by a heart rate monitor (WEP-7202, Nihon Kohden) through an analog output. Using this recorded analog output, an average value of the last 15-s period of HR was subsequently retrieved for analysis.

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Data recording.

Analog signals for EMG, pedal force, pedal positions (from four switches), HR, and NIRS sampling time (from the metronome) were simultaneously recorded by a digital recorder (RD-135T, TEAC, Tokyo, Japan) and digitized by a 16-bit analog-to-digital interface card at a sampling rate of 1 kHz, and successively stored on a desktop computer.

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Data Analysis
Normalization of NIRS values.

The present NIRS device uses a continuous wave and does not provide absolute values for oxygenation level and muscle blood volume, as the absolute path length is not determined. Consequently, changes in NIRS data expressed by the relative values of resting level were normalized for each subject. The maximal and minimal NIRS values during actual pedaling (60–220 W) were determined to be 100% and 0%, respectively. In past studies, the maximal deoxygenation level obtained by cuff ischemia was determined to be 0%. We did not use this method because it was not essential for the present purpose and causes some discomfort to the subjects.

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Determination of NIRS values at a given range of crank angles.

When the NIRS data were reordered and plotted according to the crank angle of all data (see Fig. 3), the x-values for the NIRS data were scattered across the crank angle (0–360 degrees). For statistical analysis, a representative NIRS value at each 6 degrees (0–6, 6–12, etc.; 60 sections in total) was determined by the average of values positioned in each crank angle section.

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Decision of the top/bottom peak for NIRS changes.

To clarify the relationship between changes in oxygenation level and muscle blood volume against crank angle, mathematical curve fitting to the reordered changes in both NIRS parameters was conducted with a polynomial fitting technique (Origin 6.0, Microcal, Northampton, MA). The x-values (crank angles) for top/bottom peaks of oxygenation level changes and muscle blood volume were determined as the degrees for the top/bottom of the fitted polynomial NIRS curve (see Fig. 3). The formula order of the polynomial fitting was determined by AIC theory (2). Differences in crank angles for the top/bottom peaks of oxygenation level and muscle blood volume were calculated at 180 W and 220 W work for each subject. This polynomial fitting technique was also used for the reordered changes in muscle blood volume during the Add-trial.

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Statistical Analysis

For the MAIN-trial, t-tests were performed between the means of HR at each work rate (60–220 W) and between iEMG averages at the end of the second and third minutes for each subject at each work rate. For the Add-trial, t-tests were also performed between the averages of muscle blood volume for the first and second bouts at 180 (or 210) W in each subject. Two-way analyses of variance (ANOVAs) with repeated measures were carried out to examine the effect of work rate and degree on both NIRS values obtained during the MAIN-trial. Post hoc multiple comparison using Tukey’s procedure were also conducted. For all analyses, differences were considered significant at P < 0.05.

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Physiological variables and work intensities.

V̇O2max and maximal HR (HRmax) during the Max-test were 3.51 ± 0.20 L·min−1 and 196 ± 12 bpm, respectively. HRmax for each subject was noted between −4 to +11 bpm of the corresponding age-related maximum, and the RER value ranged from 1.21 to 1.31. For each subject, V̇O2 level showed a plateau and/or leveling off at the end of the Max-test. Maximal work rate ranged from 240 to 300 W among subjects during the MAX-test. During the MAIN-trial, work rate ranged from 260 to 300 W. Statistical analyses were carried out using the values obtained below the work rate of 220 W from all subjects that completed the task for 3 min. Figure 1 shows the relationship between the iEMG averaged values at the end of the second and third minutes of the MAIN-trial, provided that the work rate was less than 220 W. There were no significant differences in values of the exercise bouts for any subjects. The relationships between mean values obtained by averaging the iEMG values and work rate not exceeding 220 W in subjects are shown in Figure 5. The iEMG values increased in a linear fashion against work rate. The means of HR at the second (105–120 s) and third minutes (165–180 s) for each exercise bout (60, 100, 140, 180, and 220 W) were 98 ± 8 and 97 ± 9 bpm, 114 ± 8 and 113 ± 9 bpm, 130 ± 8 and 132 ± 8 bpm, 152 ± 6 and 155 ± 9 bpm, and 177 ± 8 and 180 ± 9 bpm, respectively. There were no significant differences in mean values among work rates. The mean HR at the end of 220 W was 89.3 ± 4.5% of HRmax.

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Determination of crank angle for NIRS signals.

Figure 2 (top) shows some of the oxygenation changes in the NIRS signals during the MAIN-trial for a subject. Similar to the previous study for pedaling work (5) (Fig. 1), the NIRS signals changed continuously within a given range, resembling an “electromyogram.” A 3.5-s enlarged section of the NIRS signals and additional measurements such as pedal force, rectified EMG, crank position, and NIRS sampling time during the same period of exercise are also shown in Figure 2. Higher spikes for crank position indicate signals obtained at top-dead center during a crank cycle. Pedal force is positive downwards. The signal from the electric metronome, indicating NIRS sampling time, spikes every 0.5 s. The wider spike seen at every 2 s signifies the electric signal of a longer beeping sound. The relationship between NIRS sampling time and crank position determined each crank angle for the NIRS signals during a crank cycle. For example, the position for the asterisked NIRS sampling at the left end of Figure 2, between 270 and 360 (0) degrees of crank angle, is estimated as 286.5 degrees. Each crank angle for the NIRS signals during a crank cycle was obtained using the same procedures (Fig. 2, bottom).

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Reordered NIRS and biomechanical measurements.

Typical changes in muscle blood volume, oxygenation level, pedal force, averaged rectified EMG, and knee angle against crank angle for a subject during the MAIN-trial at 180 W are presented in Figure 3. The work rate corresponds to the work intensity appropriate for actual endurance cycling for this subject. The change in knee angle presented was optionally measured for some subjects to help interpret the interaction between the parameters against the crank angles. The crank angles for NIRS signals were well scattered, and the reordered changes in the NIRS signals for each subject demonstrated a series of smooth changes with small deviation against the crank angles. The reordered changes in muscle blood volume demonstrated an almost inverse relationship to the pedal force in the first half of the crank cycle. Changes in oxygenation show a similar phase comparable to muscle blood volume; however, a delay in the phase was noted. Similar differences in crank angles for the top and bottom of both NIRS parameters (A–A′ and B–B′) were observed in all the subjects. The mean values of the differences for the top/bottom at 180 W and 220 W were 26.4 ± 9.9 degrees and 27.8 ± 6.9 degrees, respectively. These are equivalent to 88 ± 32 ms and 92 ± 23 ms in time.

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Changes in the reordered NIRS at different work rates.

Figure 6 shows typical changes in muscle blood volume (panel A) and oxygenation (panel B) level for a single subject. As the work rate increased, muscle blood volume increased stepwise; however, the level was highest at 220 W. It was noted rather low at 260 W (no statistical analysis was done). Figure 4 shows the normalized reordered changes in muscle blood volume (panel A) and oxygenation level (panel B) against crank angle during the MAIN-trial. With the exclusion of the relationship between 180 W and 220 W for muscle blood volume, both NIRS parameters were found significant among work rates and crank angles. For exercise bouts performed at work rates higher than 140 W, a temporary increase (a lump) in muscle blood volume was observed at about 220 degrees. The levels of muscle blood volume at 225, 231, and 237 degrees of crank angle were significantly higher than those at 285, 291, and 297 degrees for 220 W of work rate. As for the oxygenation levels, a tendency to decrease (not significant) was also observed at crank angles from 240 to 330 degrees. The peak values of averaged pedal force increased with increasing work rate (Fig. 4C). In addition, as can be seen by the dotted line (P–Q) in Figure 4, the peaks of pedal force changes gradually shifted toward the narrower crank angle.

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Comparison of changes in muscle blood volume.

Figure 7 shows reordered changes in muscle blood volume at both exercise bouts of 180 W (subjects A–D) and 210 W (subjects E and F) during the Add-trial. The incremental exercise up to 240 (or 270) W provided a higher HR for the later exercise bout. The resulting averaged means for HR during the first and second exercise bouts were 159 ± 6 bpm and 173 ± 7 bpm, respectively. HR for the first exercise bout ranged from 74% to 83% HRmax. The muscle blood volume levels, which were obtained by calculating the average of raw data positioned between −20 and +20 degrees of the crank angle of the small peak (line a–f′ in Fig. 7), were statistically higher in the second exercise bout for each subject.

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The purpose of this study was to demonstrate changes in muscle blood volume and oxygenation level in a working muscle during a crank cycle. This study is derived from the assumption that similar circulation and oxygenation are repeated in a working muscle when stable metabolic and mechanical measures such as energy consumption, cardiac response, pedal force, and pedal cadence can be obtained at a given work rate.

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Reordering the NIRS parameters with crank angle.

In order to correctly measure the changes in muscle blood volume and oxygenation level during a crank cycle, crank angles for the sampled NIRS parameters should be well scattered in a crank cycle (0–360 degrees). Because of the maximal sampling frequency in our NIRS apparatus being 2 Hz, we adopted the cadence of 50 rpm to avoid repeated signal sampling at the same crank angles. The reason previous studies (4) adopted a cadence of 53 rpm against a sampling frequency of 1 Hz may be similar to our study. As a result, crank angles for NIRS data were well scattered (two to five data were recorded in each of the 60 crank angle sections in an individual subject); nevertheless, a reordering of the NIRS values revealed a continuous smooth change without a large deviation in NIRS values against crank angles (Fig. 3).

Poole et al. (22) reported that leg blood flows demonstrate stability and reproducibility even at considerably higher exercise intensities without stability in metabolic measurement such as oxygen consumption and blood lactate. We collected no data for gas exchange and/or O2 saturation (blood gas) during the MAIN-trial to demonstrate metabolism stability. However, no increase in HR and electric muscle activities (Fig. 1) during the time (90 s) taken for analysis of the NIRS measurements were recorded. This, we believe, enables us to assume that each change pattern for muscle blood volume and oxygenation level, which were reordered with the original NIRS signals (Fig. 2), may correctly reflect circulatory dynamics and metabolic changes in the working muscle, the vastus lateralis, against crank angles.

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Changes in NIRS measurements reordered against crank angle.

Since no previous study could be found with which to compare our results, it became difficult to interpret the changes in NIRS values against crank angles, especially the second slight decrease in muscle blood volume starting at 180–240 degrees, where no increases in muscle discharge and pedal force were noted. Comparisons of changing patterns of muscle blood volume against the same work rate at the Add-trial (Fig. 7) helped us to speculate about the circulatory dynamics in the crank cycle. We do not know whether the statistical methods were appropriate in this case; however, our observation shows that the peak level of lump of muscle blood volume beginning at 100–150 degrees was significantly higher in a duplicate bout of exercise (at 180 W or at 210 W) that was performed at a higher HR condition for each subject. This significant difference between lumps of muscle blood volume reflects a difference in muscle blood flow between the first and duplicate exercise bouts.

Assuming that blood flow restriction by muscle contraction at pedal thrusts occurs between 10 and 130 degrees of crank angle (equal to one third of a crank cycle), the duration of blood flow restriction by a pedal thrust would be 400 ms at 50 rpm. Meanwhile, the interval in heart strokes for six subjects would be from 397 ms (HR = 151) to 326 ms (HR = 184), respectively. The relationship between the duration of blood flow restriction and the HR interval predicts that 1.01 and 1.23 times of heart stroke occur during each pedal thrust; in some cases, two heart strokes occur during one pedal thrust. On the basis of these observations and calculations, we conclude that the lump of muscle blood volume demonstrated over 100–280 degrees indicates that a cluster of red blood cells, which were restricted to flow into the vascular bed because of an intramuscular pressure higher than blood pressure, abruptly entered the sampling site just after a pedal thrust.

A previous study (23) with Doppler ultrasound equipment clearly demonstrated that the femoral arterial inflow to a contracting muscle during dynamic exercise is markedly affected by transient variations in intramuscular pressure during one-leg dynamic knee extension. Restriction of the femoral arterial inflow has also been evidenced by negative blood velocities indicating a retrograde flow (31). When the relationship between intramuscular pressure and the extent of force output during isometric knee contraction (26) is considered, it is not feasible that muscle contractions for knee extension (or pedal thrust in the present study) could press and clog the femoral artery and capillaries. Therefore, the results (23) (Fig. 2) can be attributed to a restriction of blood flow in arterioles and venules. Confirmed in past studies with changes in artery blood flow velocity (23,25,31), we believe that the lump in muscle blood volume in the present study demonstrates the presence of blood flow restriction because of muscle contraction from the standpoint of circulatory dynamics in a working muscle.

As shown in Figure 3, changes in oxygenation level demonstrated a pattern similar to muscle blood volume, but with a time lag for both top and bottom peaks of these changes (presented in difference in crank angle between A and A′, and B and B′ in Fig. 3). The reason for the phase lag is unclear; however, an explanation derived from the parameters investigated might be as follows: change in muscle blood volume is directly affected by changes in intramuscular pressure because of muscle contraction and/or morphological changes in muscle during pedaling. On the other hand, the changes in oxygenation level reflect the status of aerobic energy production in mitochondria subsequent to muscle contraction. It seems reasonable that the lag between NIRS parameters observed in the present experiment reflects time lag between physical and chemical change. We have no way to confirm the validity of the lag time (about 90 ms); however, this time lag suggests that the change in oxygenation level is independent of muscle blood volume. At present, we are unable to make further comment, as we found no similar study with which to corroborate our result. Interestingly, we observed a tendency of decrease in the NIRS measurement at 240–330 degrees in the oxygenation level (Figs. 6B and 4B), whereas we did not find any increase in muscle activity and pedal force for pedaling exercise at the crank angle of 240–330 degrees. Therefore, the changes in the oxygenation level at the latter half of the crank cycle possibly indicate that a certain amount of blood passed under the probe just after pedal thrusts without being deoxygenated.

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Reordered change in NIRS measurements at different work rates.

As shown in Figures 4 and 6, the reordered changes in both NIRS parameters demonstrated differences in the level of muscle blood volume and oxygenation among different work rate conditions while maintaining the overall shape in the crank cycle. Löllgen et al. (19) have demonstrated that peak pedal force for a pedaling exercise performed at an intensity of 80% V̇O2max at 50 rpm requires at least 20% MVC of leg extension. Intensities of peak pedal forces (Fig. 4C) relative to the MVC in subjects are unclear, but taking the findings of past studies (16,24) and the change patterns in this study of muscle blood volume into consideration, it is speculated that impairment of blood flow during pedal thrust begins at work intensity between 100 and 140 W.

Furthermore, it is noted that the increase in muscle blood volume almost reached a plateau at 180 W (Fig. 4A), although pedal force (Fig. 4C) and the iEMG in the vastus lateralis muscle increased linearly until 220 W (Fig. 5). Grassi et al. (13) have reported that total Hb volume in the vastus lateralis muscle increased as a function of work up to 60–65% V̇O2max, after which it remained unchanged and/or rather decreased in some subjects. It is impossible to compare their study and the present results because the intensity of work at 180 W relative to the V̇O2max is unclear in the present study. However, the mean values of HR at the end of 180 W (155 ± 9 bpm) and HRmax (196 ± 12 bpm) during the MAIN-trial suggest that the work at 180 W corresponded to about 65–75% V̇O2max for our subjects.

Figure 6A demonstrates a trend of decrease in muscle blood volume at the end of the MAIN-trial. To avoid statistical complexity, we did not include data over 220 W (Fig. 4). However, we observed a similar decreasing trend in muscle blood volume at higher work rates in some subjects. Two explanations are possible. The first may be the excessive intramuscular pressure caused by an increase in work rate. However, this explanation may be difficult to support, since we found a similar decrease in muscle blood volume at 160–320 degrees of crank angle when intramuscular pressure should be very low because of muscle relaxation. The second explanation is an actual decrease in muscle blood volume attributable to inadequate supply by the central circulation. Andersen and Saltin (3) have pointed out that when a large fraction of a muscle mass is actively engaged in exercise such as running or cycling, the limiting factor for oxygen transport to the working muscle is not the vascular bed of that skeletal muscle but the central circulation capacity to supply blood and oxygen. Harms et al. (16) have described that increased oxygen consumption by respiratory muscle compromises blood flow to leg muscles during maximal exercise. Therefore, the decrease in blood volume in our study may reflect an actual decrease in muscle blood flow because of an increased demand of blood (O2) in the respiratory muscles and other working muscles such as triceps surae, gluteus maximus, and trunk muscles responsible for sustaining posture at about the maximal work intensity.

On the basis of the relationship between NIRS data for muscle oxygenation and femoral venous O2 saturation (blood gas) during dynamic contractions, MacDonald et al. (20) reported that NIRS does not provide a valid estimate of Hb and/or O2 saturation. Discrepancy between blood gas and NIRS measurements has also been pointed out by Costes et al. (8). However, as mentioned by MacDonald et al. (20), blood gas estimated by femoral venous blood represents the sum of all blood returning from the exercising muscles in the lower extremity. Therefore, it seems difficult to deny the validity of NIRS with discrepancy between measurements from femoral vein and a given working muscle (the vastus lateralis muscle). Moreover, as NIRS measurements reflect changes in oxygenation levels in small blood vessels (arterioles, capillaries, venules) and tissue myoglobin (7,21), so the disagreement between the NIRS measurement and blood gas originating from the femoral vein may not be a valid argument against the efficacy of NIRS.

We understand that our results do not provide further discussion concerning the validity of NIRS, as we did not measure blood chemistry. However, changes in NIRS parameters corresponded well to biomechanical measurement changes in our study (Fig. 3), and significant differences in muscle blood volume were also detected under different HR conditions (Fig. 7). Considering these factors, the present research reinforces the reliability of the NIRS signals. The NIRS technique we used here has some limitations, and there may be very few techniques available with no limitation. The NIRS device principally detects optical change at depths equal to the distance between the light source and the detector (7). Therefore, the technique is inadequate for simultaneous measurement of muscle blood volume within the whole femoral muscles. However, we consider this limitation an advantage to our original purpose—to estimate circulatory dynamics in a given muscle during pedaling exercise.

One may argue that blood flow restriction during a pedal thrust and decrease in muscle blood volume at higher work rates are not physiologically new findings by themselves. However, this is the first study that has demonstrated that changes in the NIRS parameters when reordered with crank angles show a pattern change corresponding to changes in pedal force and electrical muscle activity for pedal thrust during a crank cycle, and that the noisy changes in the NIRS signals during pedaling correctly reflect circulatory and metabolic changes in the crank cycle. In addition, this is the first study that demonstrates a temporary increase in muscle blood volume following a pedal thrust, which might occur as a result of blood flow restriction during muscle contraction for pedal thrust. On the basis of these results, we conclude that the present method of reordering the NIRS parameters against crank angle is a useful measure when investigating circulatory dynamics and metabolic changes in a working muscle during pedaling exercises.

The authors gratefully acknowledge the helpful advice from Drs. Atsuko Kagaya, Shunsaku Koga, Yoshiyuki Fukuba, Hajime Miura, Eiichi Chihara, and Yuji Morimoto, and the technological advice of Dr. Toshikazu Shiga. We also express our gratitude to Dr. Miharu Miyamura for the support throughout the process of this research; Koji Ishida for the provision of experimental devices; Hiroyuki Higuchi and Takayuki Sako for their technical advice; and Jiro Takai, Islam Mohammond Monirui, and Albert P. Dudley for their careful readings of the manuscript.

Address for correspondence: Tetsuo Takaishi, Institute of Natural Sciences, Nagoya City University, Mizuho-ku, Nagoya 467-8501, Japan; E-mail:

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