Preconditioning Effects of Neuromuscular Electrical Stimulation in Patients with Symptomatic Peripheral Arterial Disease

Abstract

Aims: Intermittent claudication is a major barrier to establishing an exercise routine in patients with peripheral artery disease (PAD). This study investigated the preconditioning effects of neuromuscular electrical stimulation (NMES) on walking capacity in patients with PAD and intermittent claudication. Additionally, it aimed to determine the optimal NMES settings and the underlying mechanisms of its effects.

Methods: A total of 15 patients with PAD (Fontaine II) participated in a crossover study. Each patient underwent 10-min sessions of NMES at two frequencies (4 and 20 Hz) and two intensities (moderate and high), plus a sham condition, before treadmill walking tests using the modified Gardner protocol.

Results: Pain-free walking distance significantly increased after a single session of 20 Hz high-intensity NMES (259.2±27.5 m; p＜0.05) relative to the sham group (201.9±23.6 m). The maximum walking distance also improved significantly after 4 and 20 Hz high-intensity NMES. The vascular endothelial function, assessed by flow-mediated dilation, was significantly enhanced following 20 Hz moderate- and high-intensity NMES, as well as 4 Hz high-intensity NMES, relative to the sham group. Additionally, lower extremity blood flow, as measured via transcutaneous partial pressure of oxygen, improved significantly in all NMES conditions. However, serum markers such as myeloperoxidase, hepatocyte growth factor, vascular endothelial growth factor, and CD34^＋/CD133^＋ progenitor cell counts did not differ significantly between the NMES and sham groups.

Conclusion: High-intensity 20 Hz NMES is an effective preconditioning strategy for instantaneously enhancing the walking capacity in patients with PAD, likely due to nitric oxide-mediated vasodilation. These findings suggest that NMES is a promising therapeutic approach for PAD rehabilitation.

Abbreviations: ABI, ankle-brachial index; CLI, critical limb ischemia; FMD, flow-mediated dilation; HGF, hepatocyte growth factor; HIF-1, hypoxia inducible factor; IC, intermittent claudication; MPO, myeloperoxidase; NMES, neuromuscular electrical stimulation; NO, nitric oxide; NRS, numerical rating scale; PAD, peripheral arterial disease; PFWD, pain-free walking distance; tcPO₂, transcutaneous oxygen pressure; VEGF, vascular endothelial growth factor

Introduction

The incidence of peripheral arterial disease (PAD) increases with age, prevalence of smoking, and incidence of diabetes mellitus, hypertension, dyslipidemia, and obesity. A meta-analysis¹⁾ estimated that PAD affects more than 200 million people worldwide, with a particularly marked increase in Asian countries. The 5-year survival rate of patients with PAD and intermittent claudication (IC) is 70%, which is equivalent to that of malignant diseases, such as breast and colorectal cancer²⁾. Cardiovascular deaths account for 75% of mortality in patients with PAD²⁾. IC reduces physical activity, worsens arteriosclerosis, and leads to cardiovascular events^{3, 4)}. Based on several randomized trials and according to recent guidelines, supervised walking exercise therapy is a highly recommended Class 1 treatment for patients with PAD, with the exception of those with critical limb ischemia (CLI)^5-11). Exercise therapy has been shown to improve muscle oxygen utilization efficiency and induce the development of angiogenesis and collateral blood channels through the action of hypoxia-inducible factor (HIF-1), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), endothelial progenitor cells, and improved blood fluidity^{3, 5)}. However, in patients with symptomatic PAD, it is difficult to develop a routine exercise habit because of IC.

In patients with PAD, neuromuscular electrical stimulation (NMES) is effective in activating muscle pumps, improving peripheral vascular blood flow, promoting angiogenesis, and reducing inflammation. A recent pilot case study reported that the pain-free walking distance (PFWD) temporarily increased in patients with PAD and IC when NMES was applied to the lower limbs before walking^12-14). In response to the results of these pilot studies, randomized controlled trials^{15, 16)} have begun to verify the effectiveness of programs involving walking exercises after NMES. However, the optimal frequency and intensity of NMES required to achieve the maximal preconditioning effect has not been explored, and its beneficial mechanisms remain unknown.

Aims

This study aimed to examine the preconditioning effect of NMES in rehabilitation of patients with PAD, determine its optimal setting, and elucidate the mechanisms underlying its beneficial effects.

Methods

Study Design and Participants

This single-center crossover study was conducted with the approval of the Ethics Committee of Dokkyo Medical University Nikko Medical Center (approval number: Nikko 21-010), and was performed in accordance with the principles of the Declaration of Helsinki. The inclusion criteria were age ≥ 20 years, Fontaine stage II PAD with ankle-brachial index (ABI) ≤ 0.9 within the previous 6 months, and the provision of written informed consent to participate in the study. The exclusion criteria were as follows: acute coronary syndrome within 3 months of the onset; CLI, presence of a pacemaker or other implanted devices; a diagnosis of COVID-19 or other serious infections; significant pulmonary disease or heart failure; a maximum walking time of ＜60 s or ＞840 s in the modified Gardner treadmill test, deemed inappropriate for study participation by the physician; and other contraindications to exercise stress testing as described in current cardiac rehabilitation guidelines.

The sample size was determined using the G*Power software program, with a significance level of 5%, 80% power, and an effect size of 0.4, based on previous studies^{13, 14)}. This calculation, designed for a two-tailed test, was conducted for a two-way repeated-measures analysis of variance (ANOVA), yielding a target of 15 participants. The efficacy of the intervention was assessed through a per-protocol analysis that included only participants who fully adhered to the study protocol. Participants were recruited from the rehabilitation patient list for PAD at Dokkyo Medical University Nikko Medical Center (Fig.1). Of the 34 participants who met the inclusion criteria, 14 were excluded due to CLI (n = 1), pacemaker implantation (n = 1), or a modified Gardner treadmill test time at baseline of ＜60 s or ＞840 s (n = 12). Five participants dropped out during the protocol, either because of hospitalization during the evaluation period (n = 1) or difficulty in continuing the evaluation (n = 4). Ultimately, 15 participants completed all evaluations, and their data were analyzed. To evaluate the treatment effect under optimal conditions in this study, a per-protocol analysis was performed. Attending physicians were advised to avoid making changes to the participants’ medication regimens during the study period wherever possible.

Fig.1. Participant selection

Participants were recruited from a list of patients undergoing rehabilitation for peripheral arterial disease (PAD). Of the 34 participants who met the inclusion criteria, 14 were excluded due to critical limb ischemia (n = 1), pacemaker implantation (n = 1), or a baseline maximum walking distance by the modified Gardner Treadmill test of ＜60 s or ＞840 s (n = 12). Five participants dropped out during the protocol due to hospitalization during the evaluation period (n = 1) or difficulty continuing the evaluation (n = 4). Finally, 15 participants completed all evaluations, and their data were analyzed.

Protocol

Each participant underwent a treadmill walking test using the modified Gardner protocol and flow-mediated dilation (FMD) of the brachial artery as baseline assessments (Fig.2). After the baseline measurements, participants were randomly assigned to groups A–E. On day 1, each NMES intervention was administered for 10 minutes following the assessment of transcutaneous oxygen pressure (tcPO₂) in the lower legs. After the NMES intervention, FMD and tcPO₂ were measured. Thereafter, the treadmill walking test was conducted using the modified Gardner protocol to assess the PFWD and MWD. After a 5-minute rest, blood samples were collected to assess the CD34^＋/CD133^＋ progenitor cell count and HGF, VEGF, and myeloperoxidase (MPO) levels. The same procedure was repeated from days 2 to 5, with an interval of at least 7 days between the interventions.

Fig.2. Study protocol

This study design was a crossover trial. Participants were randomly assigned to groups A–E after baseline measurements. Each NMES intervention was administered on day 1. The NMES intervention was administered on days 2, 3, 4, and 5, with an interval of at least 7 days between sessions.

FMD, flow-mediated dilation; HGF, hepatocyte growth factor; NMES, neuromuscular electrical stimulation; VEGF, vascular endothelial growth factor.

Neuromuscular Electrical Stimulation

We attached five belt electrodes to the abdomen and both lower extremities in the supine position (lumbar spine: 3.93×3.5 cm; thigh: 3.69×6.5 cm; ankle: 3.54×6.30 cm; HOMER ION Laboratory Co., Ltd., Tokyo, Japan), as described elsewhere¹⁷⁾. Five stimulation patterns were applied, each lasting 10 min. The stimulation frequencies were 4 Hz and 20 Hz, with intensities set at different levels: high intensity corresponded to a numerical rating scale (NRS) of approximately 8/10, moderate intensity to an NRS of 5/10, and sham condition to an NRS of 0–0.5.

Evaluation of Walking Function

PFWD (m) and MWD (m) were measured using the modified Gardner test^{3, 5, 6)} conducted by examiners blinded to the NMES settings. PFWD was defined as the distance at which the participant first perceived leg pain, whereas MWD was defined as the distance at which walking became too difficult to continue. A medical treadmill (MAT-2200; Fukuda Denshi, Tokyo, Japan) was used for the measurements.

Blood Sampling and Vascular Endothelial Function

Blood samples were collected following a gait assessment performed after 4 Hz high-intensity NMES, 20 Hz high-intensity NMES, and sham treatment. Trained technicians measured the number of CD34^＋/CD133^＋ cells by fluorescence-activated cell sorting (FACSCalibur™; BD Biosciences, Franklin Lakes, NJ, USA) with the following antibodies: anti-human CD34^＋, CD45^＋, and CD133^＋ (BD Biosciences). After gating CD45^＋ cells (a marker of nuclear cells) from whole blood, we measured the number of cells that were double-positive for CD34 and CD133, as described elsewhere^{18, 19)}. To measure the plasma levels of HGF, VEGF, and MPO, disodium EDTA-treated plasma samples were frozen at −80℃ until the assay. Concentrations of HGF (Quantikine ELISA, Human HGF Immunoassay; R&D Systems, Minneapolis, MN, USA), VEGF (Quantikine ELISA, Human VEGF Immunoassay; R&D Systems), and MPO (IDK MPO ELISA; Immunodiagnostik AG, Bensheim, Germany) were measured by an ELISA; all assays were performed in duplicate.

FMD was measured before NMES (baseline data) and immediately after NMES on the right upper arm in the supine position using a semiautomated solution (UNEX EF; UNEX Corporation, Nagoya, Japan), as described elsewhere^{20, 21)}. An occlusion cuff was wrapped around the forearm and inflated to a compression pressure of 50 mmHg over the systolic blood pressure for 5 min. In this system, the diastolic diameter of the brachial artery was semi-automatically determined, and the changes in the diameter were automatically tracked. The FMD was calculated as the percentage increase in the vessel diameter of the brachial artery following release from compression. The transcutaneous oxygen pressure (tcPO₂) monitor (TCM400; Radiometer, Copenhagen, Denmark) was calibrated according to the manufacturer’s specifications. The probe was applied to the center of the affected lower leg, and the average value for 1 min before the start of NMES was used as the pre-NMES value²²⁾. NMES was initiated after a 5 min rest period, and the average value for 1 min immediately after the NMES ended was used as the post-NMES value for comparison.

Statistical Analysis

Data are expressed as the mean±standard deviation or standard error (SE). We compared the walking distances in the intervention and sham groups using a two-way repeated measures ANOVA with Tukey’s method for post hoc testing. The number of progenitor cells expressing both CD34^＋ and CD133^＋ antigens, VEGF, and HGF was compared by a one-way ANOVA for the following three groups: 20 Hz high-intensity NMES, 4 Hz high-intensity NMES, and sham treatment. The delta values for FMD and tcPO₂ between the baseline and immediately after each NMES procedure were compared with those in the sham group using a one-way ANOVA with Tukey’s method for post hoc testing. Spearman’s rank correlation coefficient was used to evaluate the correlation between ΔFMD and ΔPFWD or ΔMWD for each NMES setting as well as between ΔtcPO₂ and ΔPFWD or ΔMWD for each NMES setting. All statistical analyses were performed using SPSS (ver. 28, IBM, Armonk, NY, USA), with significance set at p＜0.05.

Results

The mean age of the participants (n = 15 [male, n = 11; female, n = 4]) was 72.1±17.0 years (Table 1). The baseline values for performance measures were as follows: PFWD, 186.4±84.9 m; MWD, 370.7±202.8 m; and FMD, 5.0±1.5%. The comorbidities and PAD lesions are shown in Table 1. After a single session of 20 Hz high-intensity NMES, PFWD significantly increased (mean±SE, 259.2±27.5 m) relative to the sham condition (201.9±23.6 m; p = 0.040; Fig.3a). Similarly, MWD significantly improved with both 20 Hz high-intensity NMES (437.5±49.2 m; p = 0.018) and 4 Hz high-intensity NMES (449.3±50.7 m; p = 0.003) relative to the sham condition (378.6±49.0 m; Fig.3b).

Table 1.Clinical Background of Study Patients (n = 15)

Age (years)	72.1±16.6
Sex Male	11
ABI	0.61±0.1
Baseline data
Pain free walking distance (m)	186.4±84.9
Maximal walking distance (m)	370.2±202.8
Flow Mediated Dilation (%)	5.0±1.5
Comorbidities (%)
Hypertension	10 (66.7%)
Dyslipidemia	9 (60.0%)
Diabetes mellitus (type 1)	1 (6.7%)
Diabetes mellitus (type 2)	7 (46.7%)
Maintenance hemodialysis	1 (6.7%)
Previous myocardial infarction	5 (33.3%)
Previous history of PCI	5 (33.3%)
Previous Cerebrovascular disease	6 (40.0%)
Ex-smoker	4 (26.7%)
Current smoker	2 (13.3%)
Medications (%)
Statin	12 (80.0%)
Insulin	2 (13.3%)
Aspirin	5 (33.3%)
Cilostazol	5 (33.3%)
Warfarin	1 (6.7%)
DOACs	2 (13.3%)
β－blokers	2 (13.3%)
ARB	6 (40.0%)
ACEi	0 (0%)
ARNI	5 (33.3%)
SGLT2i	6 (40.0%)
Diseased lower extremity arterial lesions
Common iliac artery	5
External iliac artery	1
Superficial femoral artery	8
Below-knee arteries	7

Values for age and ABI and baseline data represent mean±standard deviation.

ABI, Ankle-brachial index; ACEi, angiotensin converting enzyme inhibitor; ARB, angiotensine receptor blocker; ARNI, angiotensine receptor- neprilysin inhibitor; DOACs, direct oral anticoagulants; PCI, percutaneous coronary intervention; SGLT2i, sodium/glucose cotransporter 2 inhibitor

Fig.3. Pain-free walking distance (PFWD) and maximum walking distance (MWD) after neuromuscular electrical stimulation (NMES)

Data are shown as the mean±SE. PFWD significantly increased immediately after 20 Hz high-intensity NMES relative to the sham condition (A). The MWD also significantly increased after 20 Hz high-intensity and 4 Hz high-intensity NMES relative to the sham condition (B). Comparisons were made using a repeated measures analysis of variance with Tukey’s post hoc test.

Mod, moderate; MWD, maximum walking distance; PFWD, pain-free walking distance.

Delta FMD values from baseline significantly improved after 20 Hz moderate-intensity (2.2±0.5%; p = 0.005; Fig.4), 20 Hz high-intensity (3.6±0.5%; p = 0.00000025), and 4 Hz high-intensity (2.9±0.4%; p = 0.000053) relative to the delta sham condition (0.5±0.5%). Delta tcPO₂ values also significantly improved from baseline after all single-session NMES settings relative to the sham condition (0.9±0.5 mmHg; Fig.5). The results showed significant differences for Δ20 Hz moderate-intensity (13.3±2.9 mmHg; p = 0.008), Δ20 Hz high-intensity (12.9±3.1 mmHg; p = 0.023), Δ4 Hz moderate-intensity (14.4±4.3 mmHg; p = 0.046), and Δ4 Hz high-intensity (15.8±3.0 mmHg; p = 0.004) relative to the delta sham condition.

Fig.4. Change from baseline in flow-mediated dilation (FMD) values after neuromuscular electrical stimulation (NMES)

Data are shown as the mean±SE. Delta FMD significantly increased at 20 Hz moderate (mod)-intensity, 20 Hz high-intensity, and 4 Hz high-intensity NMES relative to the sham condition.

Fig.5. Change from baseline in transcutaneous partial pressure of oxygen (tcPO₂) after neuromuscular electrical stimulation (NMES)

Data are shown as the mean±SE. The ΔtcPO₂ of the leg sig nificantly increased at 20 Hz moderate (mod)-intensity, 20 Hz high-intensity, 4 Hz moderate-intensity, and 4 Hz high-intensity NMES relative to the sham condition.

No correlations were found between ΔFMD and ΔPFWD from baseline (Fig.6A); however, ΔMWD and ΔFMD from baseline were weakly correlated (Fig.6B). Additionally, no correlations were observed between ΔtcPO₂ and ΔPFWD or ΔMWD from baseline (Fig.6C, D).

Fig.6. Correlation between the change in walking ability and change in flow mediated dilation (FMD) or change in transcutaneous partial pressure of oxygen (tcPO₂)

There was no correlation between ΔFMD and ΔPFWD from baseline (A). However, ΔMWD from baseline was weakly correlated with ΔFMD (B). Additionally, no correlations were observed between ΔtcPO₂ from baseline and either ΔPFWD (C) or ΔMWD (D) from baseline.

There was no significant increase in CD34^＋/CD133^＋ progenitor cell numbers or serum levels of MPO, VEGF, or HGF in either the 20 Hz or 4 Hz high-intensity NMES groups relative to the sham group (Table 2).

Table 2. Comparison of plasma levels of angiogenic factors and vascular progenitor cells immediately after high-intensity NMES (at 20 Hz and 4 Hz frequencies) for 10 min

	SHAM	20 Hz High	4 Hz High	p-value
CD34＋/CD13＋ (Event Cells)	229.8±131.1	214.8±174.6	245.3±203.3	NS
HGF (pg/mL)	2620.7±747.5	2104.1±631.9	1965.2±383.1	NS
VEGF (pg/mL)	238.6±25.2	236.0±28.2	237.0±16.6	NS
MPO (ng/mL)	3.7±1.8	4.3±2.3	3.6±1.2	NS

Data are shown as mean±standard deviation.

Angiogenic and vasodilating factors for each group were compared using one-way analysis of variance, and no significant differences were found among the groups.

HGF, hepatocyte growth factor; MPO, myeloperoxidase; NMES, neuromuscular electrical stimulation; VEGF, vascular endothelial growth factor

Discussion

This study is the first to demonstrate that a 20 Hz high-intensity NMES setting applied for 10 min is optimal for alleviating claudication symptoms in patients with PAD. Additionally, significant improvements in MWD were observed after both the 4 Hz and 20 Hz high-intensity NMES protocols. The vascular endothelial function showed marked enhancement after the application of 20 Hz moderate- and high-intensity NMES, as well as 4 Hz high-intensity NMES, relative to the sham condition. Furthermore, all NMES conditions led to a significant increase in the lower-extremity blood flow. Given that IC is a primary barrier to establishing a walking routine, NMES preconditioning shows promise as an effective rehabilitation strategy for the management of symptomatic PAD.

The underlying mechanism for the acute enhancement of PFWD observed with a single session of NMES is primarily attributed to nitric oxide (NO)-mediated vasodilation, as shown in Figs.4 and 5, with a low likelihood of involvement of VEGF, HGF, MPO, or progenitor cells. Belt-type NMES activates the muscle pump by repeatedly inducing passive contraction and relaxation of the entire lower limb muscle, including the gluteal and deep inner muscles. This comprehensive engagement of muscle groups has been supported by the findings of Numata et al., who demonstrated that positron emission tomography with ¹⁸F-fluorodeoxyglucose revealed significantly higher metabolic activity in the gluteus maximus, gluteus medius, gluteus minimus, quadriceps femoris, sartorius, and hamstrings in participants using belt-type NMES relative to the control group²³⁾. Placement of the occlusion cuff during the FMD procedure modifies the shear stress stimulus and NO dependency of the vascular dilation response. In the present study, distal cuff placement relative to the echo-imaged brachial artery resulted in a predominantly NO-mediated dilation response, whereas proximal cuff placement produced dilation that was less dependent on NO²⁴⁾. We hypothesize that the enhancement of FMD in the remote brachial artery is primarily attributable to the transient increase in blood flow induced by a wide range of skeletal muscle contractions elicited by belt-type NMES. This transient increase in blood flow likely improves the endothelial function by promoting systematic endothelial nitric oxide synthase activation and NO release via shear stress exerted on the vascular walls. Our findings are consistent with those of Ishikawa et al., who demonstrated that passive contraction of lower extremity muscles induced by belt-type NMES increases FMD in the remote brachial artery after 30 min of prolonged sedentary time²⁵⁾. These findings suggest that the mechanism by which belt-type NMES enhances the remote arterial function is likely mediated by widespread activation of the lower extremity musculature, which promotes systemic circulatory and endothelial adaptations. Although the change in the endothelial function after NMES was not correlated with the changes in PFWD, a general correlation with the changes in MWD from baseline was observed. The alleviation of claudication symptoms through NMES has been attributed to factors other than the improvement of the vascular endothelial function, such as pain relief. Moreover, the subjective nature and potential ambiguity of self-reported claudication symptoms may also contribute to this discrepancy. These factors may explain why the relationship between ΔFMD and ΔPFWD in Fig.6A exhibited a non-significant trend toward correlation. Similarly, there were no correlations between the change in leg flow reflected by tcPO₂ immediately after NMES and the changes in PFWD or MWD from baseline. Improvements in walking distance require enhanced blood flow and improvements in lower limb muscle strength and the efficiency of walking movements. Although FMD may temporarily improve with a single session of high-intensity NMES, one session alone is insufficient to address the chronic dysfunction of muscles involved in walking^{26, 27)}.

No significant increases in CD34^＋/CD133^＋ progenitor cell numbers in the blood or serum levels of MPO, VEGF, or HGF were observed in the 20 Hz and 4 Hz high-intensity NMES groups relative to the sham group. Thus, these factors may not have contributed to the preconditioning effects of a single NMES session. progenitor cells are mobilized from the bone marrow into the peripheral bloodstream to promote angiogenesis²⁸⁾. Intramuscular injections of CD34^＋ cells have been previously reported to promote neovascularization and alleviate ischemia in patients with CLI^{29, 30)} and primate models of myocardial infarction³¹⁾. Our previous results¹⁸⁾, as well as those of other investigators^32-34), demonstrate that moderate-to-intense exercise over several months can increase circulating progenitor cell numbers in patients with ischemic cardiovascular diseases. We also observed a positive correlation between CD34^＋/CD133^＋ cell counts and exercise capacity after a 3-month training period following myocardial infarction¹⁸⁾, with these cells being increasingly utilized in clinical settings. Although Nagasaka et al.³⁵⁾ reported that the local expression of VEGF and HGF in the ischemic limbs of rats increased after 5 days of high-intensity NMES, no increase in VEGF or HGF was observed after a single session of NMES. This indicates that repeated NMES over several days may be essential for increasing serum levels of VEGF and HGF. We previously demonstrated that in non-option patients with PAD and IC, intensive walking immediately after the daily administration of 3,000 units of intravenous heparin for two weeks improves MWD relative to exercise therapy alone³⁶⁾. The proposed mechanism involves temporary detachment of MPO and HGF from the vascular endothelium. The detachment of MPO from the endothelium prolongs the lifespan of NO, thereby inducing vasodilation^37-39). Additionally, HGF is thought to promote angiogenesis in a more physiological manner by transiently increasing its blood concentration and redistributing HGF to the lower limbs during subsequent walking exercises³⁶⁾.

The improvement in PFWD with high-intensity stimulation at 20 Hz suggests that NMES intensity may have a stronger preconditioning effect at higher currents. Additionally, the improvement in PFWD can serve as a positive motivator for continuing exercise therapy by reducing pain, which may contribute to the development of exercise habits and further improvement in arteriosclerosis.

Study Limitations

The present study was associated with some limitations. First, the sample size was relatively small; however, statistical power was sufficient. Second, the standard deviations for the PFWD and MWD were somewhat large owing to the relatively small sample size.

Conclusion

In conclusion, 20 Hz high-intensity NMES applied for 10 min demonstrated efficacy as a preconditioning strategy to immediately enhance walking capacity in patients with PAD, likely through NO-mediated vasodilation. These findings indicate that NMES may serve as a promising therapeutic adjunct for PAD rehabilitation.

Clinical Perspectives

Two randomized controlled trials have commenced internationally to evaluate the effectiveness of interventions combining NMES with walking exercises^{15, 16)}. In our previous study, we demonstrated that intensive walking combined with intravenous heparin administration (3,000 units) for two weeks significantly improved the maximum walking distance in patients with symptomatic PAD relative to exercise therapy alone³⁶⁾. Building on these findings, we are currently preparing to extend our study by integrating NMES, heparin, and exercise into a comprehensive treatment strategy for PAD patients. Rigorous evaluation of the safety and efficacy prior to conducting the proposed randomized controlled trial has already been performed.

Acknowledgements

The authors thank Dr. Koji Uchiyama for his appropriate statistical advice, staff of the clinical laboratory department of Dokkyo Medical University Nikko Medical Center for technical support with FMD and tcPO₂ measurement, and Mrs. Keiko Yoshizawa for administrative support. We would like to thank Editage (www.editage.jp) for the English language editing.

Funding

This research was supported by a Grant-in-Aid for Scientific Research from the Japanese Ministry of Education, Culture, Sports, Science and Technology (Grant No. 21H04290 to Yasu T).

Conflict of Interest

The authors have no conflicts of interest to declare.

Author Contribution

Each author contributed significantly to the submitted work. K Ehara, Y Tamura, H Takahashi, and T Yasu initiated this study. K Ehara, H Takahashi, M Terashima, M Takahashi, and T Tsurumi performed data collection and analysis. H Sugimura, N Otani, T Tomoe, K Kitahara, and T Sugiyama recruited participants. K Ehara prepared the first draft of this manuscript. Y Tamura, T Tsurumi, and T Yasu participated in the data interpretation and manuscript revision.

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