Non-Invasive Muscle Metabolism Assessment with Near-Infrared Spectroscopy and Electrical Muscle Stimulation

Abstract

Background: Muscle biopsy, the gold standard for assessing muscle aerobic capacity, is an invasive procedure. Noninvasive alternatives, such as 31-phosphorus magnetic resonance spectroscopy (³¹P-MRS) and near-infrared spectroscopy (NIRS), provide valuable insights, with NIRS providing superior time resolution and ease of use compared with ³¹P-MRS. Objectives: This study aimed to evaluate muscle metabolism using a 6 s muscle contraction phase with electrical muscle stimulation (EMS) and to assess the impact of EMS on exercise performance under hyperbaric conditions with elevated oxygen pressure. Methods: This study included nine male participants (21 ± 2 years) who underwent 5 min of EMS on the forearm flexor muscle group, with muscle oxygen dynamics assessed using NIRS. For statistical analysis, the mean values between groups were assessed using paired t-tests, and associations were evaluated using Pearson’s correlation coefficient. Results: Spontaneous blood flow interruption during EMS-induced muscle activity indicated the potential for assessing muscle metabolism without disrupting external blood flow. A significant negative correlation was observed between oxygen consumption and changes in oxygenated hemoglobin levels during muscle activity under hyperbaric conditions. Conclusions: This study demonstrates that skeletal muscle metabolism can be measured using a brief 6 s quantitative EMS. Furthermore, hyperbaric exercise appears to enhance aerobic capacity by increasing the rate and availability of oxygen consumption during exercise.

1. Introduction

Various loads applied to muscles alter muscle hemodynamics, specifically affecting muscle oxygen dynamics. Muscle biopsy is one of the gold standards for the in vivo measurement of muscle aerobic capacity [1,2,3,4]. However, muscle biopsy is an invasive technique and therefore unsuitable for application in clinical conditions. 31-phosphorus magnetic resonance spectroscopy (³¹P-MRS) can noninvasively assess muscle metabolism by measuring phosphocreatine levels in skeletal muscle. However, the use of ³¹P-MRS is often limited due to the high cost and large size of the equipment.

Although NIRS is primarily employed to measure cerebrovascular oxygen dynamics, its practical application in muscle remains limited and not well understood. Laser light in the near-infrared (NIR) region of approximately 750–900 nm has been used to monitor the oxygen balance in vivo by utilizing its ability to be absorbed by oxygenated and deoxygenated hemoglobin in the blood. Importantly, NIRS can noninvasively measure tissue oxygen dynamics with high time resolution, capitalizing on the different absorption coefficients of oxygenated and deoxygenated hemoglobin. However, as the human body continuously balances simultaneous oxygen supply and consumption, distinguishing whether a decrease in oxygenated hemoglobin levels results from increased oxygen consumption or reduced oxygen supply is challenging. Therefore, previous studies [5,6] have reported that the temporary blocking of arterial blood flow with a pneumatic tourniquet allows for the isolation of oxygen consumption from oxygen supply. When used in conjunction with NIRS, this method enables the precise measurement of muscle oxygen utilization independent of blood flow [7].

Several studies have been conducted to clarify muscle metabolism using NIRS, starting with Chance et al. [8] and followed by McCully et al. [9], Hamaoka et al. [10], and Higuchi et al. [11]. Hamaoka et al. [7] significantly advanced the field by applying temporary arterial blood flow occlusion to accurately measure oxygen consumption using NIRS. Furthermore, Hamaoka et al. [12] successfully measured the decrease in creatine phosphate and oxygen consumption without the need for temporary arterial blood flow occlusion, which poses a significant burden on participants, by simulating blood flow occlusion through increased intramuscular pressure during a 10 s exercise phase.

To translate these research findings into practical applications, it is essential to measure oxygen metabolism in muscle with shorter exercise durations and quantitative muscle stimulation. Electrical muscle stimulation (EMS) has the potential to offer two significant advantages for measuring muscle metabolism by delivering instantaneous quantitative electrical stimulation to the muscles. First, electrical stimulation can cause an increase in intramuscular pressure, leading to a temporary state like arterial blood flow occlusion, thereby eliminating the need for surgical arterial occlusion devices and ensuring a safer measurement process. Second, EMS can induce instantaneous and quantifiable muscle contractions more effectively than voluntary contractions, resulting in more accurate and less noisy measurement data. Therefore, the first objective of this study is to determine whether it is possible to measure muscle metabolism with quantitative EMS.

Additionally, in recent years, hyperbaric therapy has gained popularity among many people to promote rapid recovery from sports injuries and reduce training fatigue [13,14]. This therapy is typically administered at mild pressures of around 1.3 atmospheres for 60 to 90 min. However, it remains unclear whether hyperbaric therapy contributes to improved physical performance [15,16,17,18,19]. Therefore, the second objective of this study is to assess whether EMS enhances exercise performance under hyperbaric conditions, where increased oxygen partial pressure is believed to improve performance, utilizing the simplicity of muscle metabolism assessment based on EMS.

2. Materials and Methods

2.1. Participants

All the experimental procedures were approved by the Human Research Ethics Committee of Fukuoka University (approval number 23-06-02) and conforming to the principles outlined in the Declaration of Helsinki.

Nine healthy men who consented to the study were included [age: 21 ± 2 (average ± standard deviation) years; body mass index: 24.5 ± 2.8 kg·m⁻²]. The participants selected for this study were athletes aged at least 19 years who adhered to a professional exercise regimen of at least 5 days per week. Individuals with chronic cardiac, ear, nose, throat, psychiatric, or orthopedic conditions were excluded. Individuals who regularly used medication for heart disease or steroid preparations were excluded. All participants received comprehensive information regarding the purpose, content, and procedures of the experiment, and written informed consent was obtained. One of the nine participants ceased the experiment immediately and withdrew from participation because of ear pain experienced within the hyperbaric capsule. Consequently, the final number of participants available for the analysis was eight.

2.2. Study Design

The measurements were conducted at the Faculty of Sports and Health Science Clinic, Fukuoka University. The experiment was conducted at the Sports Medicine Laboratory of the Faculty of Sports Science at Fukuoka University. The participants were placed in a sitting rest position for 15 min after entering the laboratory. Thereafter, they were placed in a supine position on a bed, a muscle oxygen saturation (SmO₂) measurement probe (near-infrared spectroscopy [NIRS]) of approximately 5 cm was attached to the test muscle, and gel pads for EMS energization were attached to both sides.

The near-infrared spectrometer for SmO₂ measurements was activated and a 5 min resting measurement was performed. EMS energization was applied for 5 min to induce involuntary contractions in the test muscle. After measurement during muscle contraction was completed, a 20 min recovery measurement was performed. All measurements were completed within 30 min: at rest (5 min), during muscle contraction (5 min), and during recovery (20 min). At the end of the test, the measuring device was removed, and the participant was allowed to leave the room after their physical condition was checked. These tests were performed under two conditions: atmospheric (1.0 atm) and hyperbaric (1.3 atm).

2.2.1. Near-Infrared Spatially Resolved Spectroscopy

The NIR spatially resolved spectroscopy (NIR_SRS) probe consisted of one light source and two photodiode detectors, with optode distances of 20 mm and 30 mm. The measurements were taken using a dual-wavelength (770 and 830 nm) light-emitting diode near-infrared spatially resolved spectroscope (NIR_SRS; Hb10, ASTEM, Kanagawa, Japan). The NIR_SRS technique provides continuous, non-invasive monitoring of changes in oxygenated (oxHb), deoxygenated (dxHb), and total hemoglobin (toHb) concentrations.

2.2.2. Measurement of Subcutaneous Fat Thickness for NIRS

The thickness of the fat layer greatly affects NIR_SRS variables when the NIR_SRS technique is used [20]. A previous review suggested that NIRS data can be corrected using the thickness of the fat layer [21]. Thus, the thickness of the fat layer at the measurement site was assessed using an ultrasound device (SonoSite iViZ, FUJIFILM Medical Co., Ltd., Tokyo, Japan) before each trial, and ΔoxHb, ΔdxHb, ΔtoHb, and SmO₂ were calculated using fat-correction software (vc_oxi Version 1.0, ASTEM, Kanagawa, Japan).

2.3. Electrical Muscle Stimulation

Quantitative muscle contraction–relaxation movements were generated electrically to observe oxygen consumption and reoxygenation in the test muscle. Involuntary muscle contraction exercises were performed because it is extremely difficult to define the amount of exercise that can be performed using voluntary muscle contractions. EMS energization pads (Sakai Medical Co., Ltd., Tokyo, Japan) were applied to the forearm flexor muscle group on both sides of the NIRS device. Electrical stimulation volume was 25 volt, 30 Hz, and the electrical stimulation duration was 6 s (1.5 s ascending, 3.0 s continuous, and 1.5 s descending). Energization (6 s) and no stimulation (3 s) were repeated intermittently in 9 s cycles for 5 min. The 25-volt energization is a voltage commonly used for other dynamic strength training of athletes and for improving intramuscular blood circulation. The participant felt a strong contraction of the subcutaneous muscle group, although the sensation of electrical stimulation varied with skin resistance. In the unlikely event that a participant felt strong pain during energization and complained that the test could not be continued, the test was stopped immediately (note that no health problems such as muscle damage have ever been reported at this level of energizing dose and duration).

2.4. Measurement of Muscle Oxygen Consumption

Muscle oxygen consumption was determined from the phases of plateauing toHb and decreasing oxHb during each muscle contraction during a 5 min exercise.

Specifically, muscle oxygen consumption was calculated from the decrease in oxHb concentration per second. This calculation was performed during the phase when toHb concentration was assumed to be maintained at a constant level due to the increase in intramuscular pressure.

2.5. Measuring Conditions

The same experiments below were carried out under atmospheric and hyperbaric conditions.

The participant was placed in the supine position in an oxygen chamber with the NIRS attached to the forearm muscle group, and the EMS was placed proximally and distally between the NIRS. In the hyperbaric condition, the chamber hatch was closed, and resting measurements (5 min) were started when the pressure reached 1.3 atm from the start of pressurization, after which the EMS was energized. Measurements in the atmospheric and hyperbaric environments were performed on separate days. The degree of pressurization was measured using a barometer located in the chamber. The duration of pressurization was 30 min, which is shorter than the duration of use of a typical oxygen capsule (60–90 min). For safety reasons, the air pressure in the chamber was not increased above 1.3 atm. The oxygen partial pressures were estimated to be 160 mmHg and 207 mmHg under atmospheric and hyperbaric environmental conditions, respectively.

2.6. Statistical Analysis

The sample size was calculated considering type 1 and type 2 errors based on the statistical testing of muscle oxygen consumption, which is one of the primary outcomes. No previous studies have evaluated the effect of muscle metabolism with electrical muscle stimulation. Therefore, we have calculated the sample size using the G*Power software (Version 3.1; Bonn University, Bonn, Germany), based on a previous study by Eiken et al. that evaluated differences in knee extension torque under three conditions: an atmospheric-pressure environment (1 ATA), a high-pressure environment (1.3 ATA), and an ultrahigh-pressure environment (6 ATA) [22]. In this setting, the sample size was calculated at 80% power and a 5% significance level. This resulted in a sample size of eight subjects per group. Therefore, we decided to enroll nine subjects per group in case of dropouts.

While non-parametric tests are often suitable for small datasets, in this study, we conducted the Shapiro–Wilk test, which is widely considered one of the most appropriate normality tests for small sample sizes. The results indicated that all variables used in the analysis significantly met the assumption of normality. Additionally, to further verify the validity of the normality assumption, we generated quantile–quantile (Q-Q) plots and calculated 95% confidence intervals. Based on these results, we determined that the data in this study adhered to normality and thus applied parametric tests, including the paired t-test and Pearson product-moment correlation coefficient.

Data are presented as means and 95% confidence intervals. Differences in mean values between the two conditions were analyzed using a paired t-test. The Pearson product-moment correlation coefficient was used to analyze the correlations between the two factors. All statistical analyses were performed using SPSS Statistics 29 (IBM SPSS Japan, Tokyo, Japan), and the significance level was set at p < 0.05.

3. Results

Eight participants did not experience any problems after the experiment. The results are presented in Figure 1, with case numbers omitted from this figure and graph to protect personal data.

A typical example of muscle oxygen dynamics during 6 s contractions and 3 s relaxations with EMS of the skeletal muscle is shown in Figure 1.

The increase in intramuscular pressure caused by 6 s contractions due to EMS resulted in a steady state of toHb, which indicates the blood volume of skeletal muscle, even without arterial blood flow occlusion, suggesting the possibility of measuring oxygen consumption in skeletal muscles under atmospheric conditions (Figure 2).

It was also suggested that in hyperbaric conditions (1.3 atm), as in atmospheric conditions, the increase in intramuscular pressure caused by the 6 s contraction by EMS may result in a steady state of toHb in the skeletal muscle, and the possibility of measuring the oxygen consumption of skeletal muscle without arterial blood flow occlusion (Figure 2).

A comparison of muscle oxygen consumption, which indicates the speed of muscle oxygen consumption during blood flow occlusion caused by increased intramuscular pressure between atmospheric and hyperbaric conditions, confirmed a higher trend under hyperbaric conditions (Figure 3a). In contrast, muscle oxygen recovery was comparable between the two groups (Figure 3b).

No significant correlation was identified between muscle oxygen consumption and changes in oxHb levels during EMS under atmospheric conditions (Figure 4a). In contrast, a significant negative correlation was identified between muscle oxygen consumption and changes in oxHb levels during EMS under hyperbaric conditions (Figure 4b).

A positive correlation trend was observed between changes in oxHb levels during EMS and muscle oxygen recovery under atmospheric conditions (Figure 5a). In contrast, no significant association was identified between muscle oxygen recovery and changes in oxHb levels during EMS under hyperbaric conditions (Figure 5b).

4. Discussion

Temporary arterial blood flow occlusion is considered the gold standard for assessing muscle oxygen kinetics and the balance between oxygen supply and consumption during EMS; however, whether EMS can serve as a substitute was unclear. Therefore, this study investigated whether muscle metabolism could be measured using 6 s of muscle stimulation with EMS. In addition, we evaluated whether quantitative muscle stimulation with EMS improves exercise performance under hyperbaric conditions with elevated oxygen pressure.

The main results of this study are as follows. (i) Spontaneous blood flow occlusion occurred during 6 s contractions due to EMS in both atmospheric and hyperbaric conditions, allowing for the measurement of skeletal muscle oxygen consumption. (ii) Muscle oxygen consumption during EMS tended to be higher under hyperbaric conditions than under atmospheric pressure. (iii) Under atmospheric conditions, no significant correlation was observed between muscle oxygen consumption and oxHb changes, but a positive correlation trend was observed with the recovery rate. Conversely, in hyperbaric conditions, a significant negative correlation was observed with muscle oxygen consumption, but no association with muscle oxygen recovery. These results suggest that spontaneous blood flow occlusion occurred during EMS-induced muscle activity, which may allow muscle metabolism to be assessed without external blood flow occlusion by the combined use of NIRS and EMS.

An arterial blood flow occluder is a medical device used during surgical procedures, but it is difficult for the public to use. The findings of this study suggest that it is possible to measure muscle metabolism using EMS, which is widely available for public use, rather than relying on surgical medical devices. The method of measuring muscle aerobic capacity by the combined use of NIRS and EMS does not require the external occlusion of the arterial blood flow, which significantly reduces the burden on the participants. In addition, because the method is simple and can be used anywhere, it may be applied to areas such as the lower back and upper arm where the occlusion of the arterial blood flow with a cuff is difficult. Furthermore, because the device is small and simple, it can be implemented in the field, and the ripple effect in this study was estimated to be high.

On the other hand, there was no significant difference in muscle oxygen consumption between the atmospheric and hyperbaric conditions. This observation may be attributed to the fact that the low pressure of hyperbaric conditions (1.3 atm) did not allow enough oxygen to dissolve in the blood, thus preventing the benefits of hyperbaric conditions from being enjoyed. Cabrić, et al. [23] reported a 4.4–10% increase in maximal oxygen uptake in a study using a medical oxygen capsule capable of reproducing high-pressure (up to 2.0–2.8 atm) and hyper-oxygenated (up to 100%) conditions. Furthermore, several studies have investigated the effects of inhaling oxygen at concentrations of 92.5–100% under pressures of 1.3–3.95 atm. Pirnay et al. [24] reported that VO₂ max during exercise under 100% normobaric hyperoxic conditions was approximately 3% higher than that during normobaric air breathing. However, VO₂ max during hyperbaric oxygen breathing was similar to that under normobaric hyperoxic conditions, with no significant increase observed. The prolonged time-to-exhaustion during hyperbaric oxygen exposure was not statistically significant [25,26]. Previous studies have reported that muscles exhibited greater force per torque and higher total average power during hyperbaric oxygen exposure than during normobaric air conditions [22,27,28]. Acute exposure to hyperbaric oxygen during exercise resulted in a reduced stroke volume [29], while the heart rate remained unchanged or decreased [22,24,26,29,30,31], and cardiac output was reduced [31]. Several studies have shown that hyperbaric oxygen exposure decreases muscle blood flow during exercise and increases mean arterial pressure compared to normobaric air and 7.4% hyperbaric oxygen [30,31]. Furthermore, post-exercise muscle blood flow was reported to be lower under hyperbaric oxygen conditions than under normobaric and hyperbaric air conditions [32]. Maximal ventilation [24], alveolar ventilation [33], and blood lactate concentration [24,34] were lower during exercise in hyperbaric oxygen than during exercise in normobaric air. In their review, Šet et al. [35] reported that Banister et al. [36] and Taunton et al. [37] observed lower heart rate and ventilation and higher estimated VO₂ in hyperbaric oxygen; however, the representativeness of these results is questionable because of the small sample size in each study, typically only two. Additionally, Labanca et al. reported that joint instability can lead to abnormalities in muscle strength, highlighting the need for a multifaceted approach when assessing exercise performance [38].

In summary, a tendency was observed for muscle oxygen consumption to be greater in hyperbaric conditions than in atmospheric-pressure conditions (Figure 3a). Furthermore, a significant negative correlation was found between muscle oxygen consumption and changes in oxHb during electrical muscle stimulation, but only in hyperbaric environments (Figure 4b). This indicates that exercising in high-pressure settings may enhance the muscles’ ability to utilize oxygen more efficiently, potentially leading to improved exercise performance. Consequently, if high-level training in such environments becomes feasible, it may also enhance exercise performance in atmospheric-pressure conditions.

In the future, if measurements are performed using medical oxygen capsules that can reproduce hyperbaric and hyperoxic environments, significant changes may be observed. However, as Moriguchi et al. [39] reported, even in a high-pressure environment of 1.3 atm, cases have been reported in which patients complained of earache and had to discontinue use, so the choice must be made in consideration of the balance between benefits and burden.

The fact that higher oxygen consumption in a high-pressure environment correlates with greater change in oxHb levels suggests that individuals with a faster rate of oxygen uptake into the tissues can consume more oxygen. One possible reason for the pronounced results observed only under the high-pressure condition could be the slight increase in the amount of dissolved oxygen in the blood at rest under high-pressure conditions, which increased the amount of oxygen available to the muscle and may have contributed to this result. Eiken et al. [22] reported that the rate of decrease in muscle exertion was lower under high-pressure conditions than under atmospheric-pressure conditions during continued knee extension exercises. Furthermore, Takezawa et al. [28] reported that hyperbaric and hyperoxic environments resulted in a higher average total workload and no decrease in peak rotational speed during cycle ergometer exercise compared with normobaric and normoxic environments. However, regarding the positive correlation trend between the rate of muscle oxygen recovery and the change in oxHb only under atmospheric conditions, it is possible that oxHb recovered the amount consumed due to biological homeostasis. Therefore, there was no significant difference between the rate of muscle oxygen recovery and the change in oxHb under hyperbaric conditions, where the partial pressure of oxygen is higher than atmospheric pressure.

The limitations of the study include, first, the relatively small sample size of the participants. The accuracy of statistical analyses increases with larger sample sizes; however, investigating large samples requires substantial time and resources, making it impractical. As a result, many studies rely on small samples to infer population characteristics. In this study, we addressed the limitations of a small sample size by presenting a 95% confidence interval for all figures. Future research should focus on increasing the sample size while considering how various errors may affect the reliability of the conclusions.

Secondly, individual variability in muscle stimulation response should be noted. Due to the nature of EMS, there may be limitations arising from the fact that fat layers are less conductive to electricity. Although the same amount of electrical muscle stimulation was applied to all participants, it is not clear whether all participants experienced the same muscle contractions. Future studies need to quantify the degree of muscle contractions using electromyography.

Thirdly, the limitations of NIR technology should be considered. While NIRS can non-invasively collect information from within the body, it may be influenced by surface characteristics (e.g., skin condition, subcutaneous fat thickness, and blood Hb concentration). In this study, we recruited participants with similar subcutaneous fat thickness and analyzed changes rather than raw data to maximize the exclusion of individual variability effects. In future research, it is important to use devices that can calculate absolute values for individual comparisons, such as an NIR time-resolved spectroscope, and to combine them with other equipment to mitigate the limitations of NIRS technology.

Addressing these issues is expected to further develop the findings of this study and increase its applicability in sports practice and rehabilitation.

5. Conclusions

Skeletal muscle metabolism was measurable with a shorter quantitative stimulation using EMS (6 s). Furthermore, exercise under hyperbaric conditions could be one of the exercise methods to improve the aerobic capacity of skeletal muscles, as the rate of oxygen consumption during EMS is faster and more oxygen is available.

For the public, using EMS in a hyperbaric environment may achieve effects comparable to those in atmospheric-pressure conditions with lower-intensity muscle activity. Similarly, for athletes, utilizing EMS in hyperbaric environments could enhance exercise performance beyond what is achievable in atmospheric conditions. Consequently, this approach may facilitate muscle activity that is not possible in atmospheric-pressure settings, thereby slightly exceeding the limits of exercise performance.