Using near‐infrared spectroscopy to investigate the effects of pressures and durations of cupping therapy on muscle blood volume and oxygenation

Cupping therapy has been widely used to manage musculoskeletal impairment. However, the effects of pressure and duration of cupping therapy on the hemodynamic activity of the muscle have not been investigated. A 2 × 2 repeated measures factorial design was used to examine the main effect and interaction of pressure (−225 and −300 mmHg) and duration (5 and 10 min) on biceps muscle blood flow using near‐infrared spectroscopy in 18 participants. The results showed that a significant interaction is between pressure and duration on deoxy‐hemoglobin (p = 0.045). A significant main effect of pressure is on oxyhemoglobin (p = 0.005) and a significant main effect of duration is on oxyhemoglobin (p = 0.005). Cupping therapy at −300 mmHg for 10 min results in a higher oxyhemoglobin (6.75 ± 2.08 μM) and deoxy‐hemoglobin (1.71 ± 0.78 μM) compared to other three combinations. Our study provides first evidence that the pressure and duration factors of cupping therapy can significantly affect muscle blood volume and oxygenation.

However, the effects of pressure and duration of cupping therapy on the hemodynamic activity of the muscle have not been investigated. A 2 Â 2 repeated measures factorial design was used to examine the main effect and interaction of pressure (À225 and À300 mmHg) and duration (5 and 10 min) on biceps muscle blood flow using near-infrared spectroscopy in 18 participants. The results showed that a significant interaction is between pressure and duration on deoxy-hemoglobin (p = 0.045). A significant main effect of pressure is on oxyhemoglobin (p = 0.005) and a significant main effect of duration is on oxyhemoglobin (p = 0.005). Cupping therapy at À300 mmHg for 10 min results in a higher oxyhemoglobin (6.75 ± 2.08 μM) and deoxy-hemoglobin (1.71 ± 0.78 μM) compared to other three combinations. Our study provides first evidence that the pressure and duration factors of cupping therapy can significantly affect muscle blood volume and oxygenation.

K E Y W O R D S
cupping therapy, dose, efficacy, muscle, near infrared spectroscopy, negative pressure

| INTRODUCTION
Cupping therapy is performed by applying cups on the selected skin locations or acupoints for creating negative pressure by either fire or suction [1]. The application of negative pressure to soft tissue has been used to manage various musculoskeletal disorders [2]. Clinically, evidence to support cupping therapy on treating diseases remains insufficient [3]. The lack of dose-response relationship of cupping therapy may limit the effectiveness of cupping therapy [3,4]. The application of an effective cupping intervention requires an appropriate selection of the cupping duration and cupping pressure [2,5,6].
Various physiological mechanisms have been proposed to explain potential benefits of cupping therapy [2,7,8]. The mechanical effect of cupping therapy may stretch underlying tissue and result in an increase in local blood flow [8]. Physical stress (tension on soft tissue inside the cup and compression on soft tissue under the rim of the cup) induced by cupping therapy causes a vasodilatory response, leading to the appearance of cupping marks [9]. The microcirculatory system of the skin and muscle provides nutrients and oxygen to cells and removes metabolic wastes. The increase in local blood flow may contribute to the healing effect of cupping therapy [2,10]. However, there are no studies assessing the effect of various durations and pressures of cupping therapy on muscle blood flow that is an essential to establish the dose-response relationship of cupping therapy on improving muscle health and function [2,5,6].
Near-infrared spectroscopy (NIRS) provides noninvasive and continuous monitoring of tissue oxygenation [11,12]. NIRS has been used to assess dynamic changes in tissue oxyhemoglobin (HbO 2 ), deoxy-hemoglobin (Hb), total blood volume (BV) and oxygenation of the muscle [11]. Because of its ability to monitor blood volume and oxygenation to the local tissue, this technology may be a promising tool to investigate the dose-response relationship of cupping therapy to improve blood volume and oxygenation of the muscle [13][14][15]. Gao et al. used NIRS to investigate the therapeutic effect of cupping therapy and found a significant increase in deoxyhemoglobin and a significant decrease in oxyhemoglobin in the tissue after cupping therapy [14]. Kim et al. embedded a NIRS sensor into the cupping cup and showed that oxyhemoglobin decreased and deoxyhemoglobin increased after cupping therapy [15]. These findings provide initial evidence of cupping therapy on improving blood volume and oxygenation of the muscle. However, the effects of various pressures and durations of cupping therapy on the hemodynamic response of the muscle have not been investigated.
Under different negative pressures, cupping therapy can cause different responses of muscle blood flow that may affect efficacy of cupping therapy on treating musculoskeletal impairment [5,6]. Although there is no study investigating how various pressures of cupping therapy could affect hemodynamic responses of the muscle, the response of skin blood flow to cupping therapy could be used as a reference to estimate muscle responses. Wang et al. demonstrated that À300 mmHg was more effective on increasing skin blood flow compared to À225 mmHg using laser Doppler flowmetry [5]. Although the study of Wang and colleagues was conducted on the skin, their finding may imply that insufficient negative pressure may not be able to induce a homonymic response, including the circulatory system of the muscle and skin. Huber et al. showed that senior practitioners were able to provide negative pressure around À300 mmHg, while junior practitioners could not provide constant negative pressure [16]. Their finding may imply that various pressures may be applied in clinical practice; and when the applied pressure is insufficient, the efficacy of cupping therapy may not be achieved. Overall, the influences of applied negative pressures of cupping therapy on muscle hemodynamic responses as well as treatment efficacy remain largely unknown [17].
The duration of cupping therapy could be another important factor on modulating muscle blood flow responses. Tehseen et al. demonstrated that a longer duration (i.e., 12 min) was more effective on alleviating neck pain compared to a shorter duration (i.e., 8 and 10 min) [18]. Bummo and Soobyeong demonstrated that when cupping pressure is at À600 mmHg, the cupping duration should not be more than 200 s [19]. Wang and colleagues demonstrated that cupping therapy with various durations of negative pressure may cause different skin blood flow responses [5]. However, Wang et al. did not investigate the effects of cupping durations on muscle blood flow responses. Up to date, the duration factor of the doseresponse relationship of cupping therapy on muscle blood volume and oxygenation remains largely unknown.
The goal of this study was to investigate the effect of various pressures and durations of cupping therapy on muscle blood volume and oxygenation. The aims of this study were to: (1) investigate the main effect of pressure and duration factors of cupping therapy on muscle hemodynamic responses, including oxyhemoglobin, deoxyhemoglobin, blood volume and oxygenation, and (2) examine whether an interaction exists between the pressure and duration factors of cupping therapy on muscle hemodynamic responses, including oxyhemoglobin, deoxyhemoglobin, blood volume and oxygenation. To the best of our knowledge, this is the first study investigating the effect of various pressures and durations of cupping therapy on muscle blood volume and oxygenation responses.

| METHODS
A 2 Â 2 factorial design with repeated-measures was used to investigate the main effect of pressure and duration factors and the interaction effect between the pressure and duration factors of cupping therapy. The use of repeated measures design allowed each subject to participate in all four protocols for a higher statistical power in this study. The pressure factor included two levels at À225 and À300 mmHg and the duration factor included two levels at 5 and 10 min. The selection of these values of cupping pressure and duration were based on common combinations in clinical practice [5,20]. The applied negative pressure usually ranges from À225 to À300 mmHg with a duration from 5 to 10 min [5,20]. Each participant completed four protocols including (A) À225 mmHg for 5 min, (B) À225 mmHg for 10 min, (C) À300 mmHg for 5 min, and (D) À300 mmHg for 10 min. The counterbalanced design was implemented to minimize the order effect of 4 cupping protocols conducted in 4 different days. The testing order of four protocols for all participants is shown in Table 1. This study was approved by the University Institutional Review Board of the University of Illinois at Urbana-Champaign (IRB #22900). All participants gave written consent before participating in this study.

| PARTICIPANTS
The inclusion criteria included age between 18 and 40 years, no open wounds, scar or non-blanchable response of the skin over the biceps and the triceps of the dominant side, no diagnosed cardiovascular diseases, no diabetes, and no smoking history. Eighteen participants (12 females, 6 males) met the criteria and were recruited into this study. Their characteristics were (mean ± SD): age 24.4 ± 4.7 years, height 159.8 ± 27.3 cm, weight 69.9 ± 27.5 kg, systolic blood pressure 106.2 ± 9.7 mmHg, diastolic blood pressure 62.7 ± 8.0 mmHg, arm length 32.9 ± 2.2 cm, and arm circumference 27.4 ± 3.6 cm.

| INSTRUMENTATION
An electronic cupping therapy device (P1000-PCS, Medical Device Manufacturing Facility, CA) was used to produce negative pressure inside the cupping cup. The intensity of cupping therapy can be adjusted by setting the value at a specific value of negative pressure of this electronic cupping device ( Figure 1). This type of cupping is usually refereed as dry cupping. The cup with the 45-mm diameter (the inner diameter as 45 mm and the outer diameter as 53-55 mm) was used in this study. The selection of this cup size was based on the commonly The specific test orders of four cupping therapy protocols, including (A) À225 mmHg Â 5 min, (B) À225 mmHg Â 10 min, (C) À300 mmHg Â 5 min, and (D) À300 mmHg Â 10 min for the first 6 participants. The test orders for the rest 12 participants followed the same principle of counter-balanced design to minimize the order effect.

Participants
The order of four protocols used sizes of the cup in clinical practice [5,6]. The site for cupping therapy was chosen at the biceps (biceps belly and one third of the length from the cubital fossa to the acromion) of the dominant arm [21]. The fNIRS device (fNIR Imager 1000, fNIR Devices, LLC, Potomac, MD) was used to monitor changes in hemodynamic activity of the biceps muscle, including oxyhemoglobin (μM), deoxy-hemoglobin (μM), blood volume (μM) and oxygenation (μM). The fNIRS sensor band consisted of 10 photodetectors and 4 LED light sources with a 2.5-cm distance between a source and a detector for a total of 16-channel signals [22,23]. In this study, fNIRS signals from the four channels were averaged to represent the changes in muscle hemodynamic responses inside the cup. The oxygenated form of hemoglobin (oxyhemoglobin) has a peak absorption at 850-900 nm, and the deoxygenated form of hemoglobin (deoxy-hemoglobin) has a peak absorption at 730-750 nm [24]. The detection depth was about 1.25 cm [25]. The concentration changes (post-cupping-pre-cupping muscle hemodynamic responses) in oxyhemoglobin (Δ HbO 2 ½ ), deoxyhemoglobin (Δ Hb ½ ), blood volume (Δ HbO 2 ½ þΔ Hb ½ ), and oxygenation (Δ HbO 2 ½ À Δ Hb ½ ) were reported in this study [22]. The fNIRS signals were sampled at 2 Hz during 5-min pre-cupping and 10-min post-cupping. The spectroscopy device settings were modified depending on the participant to maintain infrared light-emitting diode intensities within a functional range that avoided dark noise levels and saturation. The raw fNIRS signals were low-pass filtered with a finite impulse response filter of cut-off frequency at 0.14 Hz to eliminate possible respiration and heart rate signals and unwanted high frequency noise [22,23]. The fNIRS signals of 5-min pre-cupping period was used to calculate the relative change of fNIRS signals in the concentration of oxyhemoglobin and deoxy-hemoglobin. In this study, only channels from 9, 10, 11, and 12 (area inside the cupping area) were used to assess hemodynamic responses of the biceps.

| EXPERIMENTAL PROCEDURES
All examinations were performed in a research lab at the University of Illinois at Urbana-Champaign. The room temperature was maintained at 24-26 C throughout the experiment. Participants were asked to be in a supine position for at least 30 min to acclimate to the room temperature. The participant fully extended the elbow with the palm facing upward of the dominant hand. The angle between the arm and the body was 45 while in the supine position. The participant was instructed to relax without any unnecessary muscle contraction. A mark was drawn on the biceps muscle at the location of onethird of the distance between the cubital fossa to the acromion. Pre-cupping measurements of fNIRS signals were 5 min and post-cupping measurements of fNIRS signals were 10 min. In order to minimize the influence of environmental light on fNIRS measurements, a compression bandage was used to wrap around the fNIRS sensor to the arm. Four cupping protocols (i.e., (A) À225 mmHg for 5 min, (B) À225 mmHg for 10 min, (C) À300 mmHg for 5 min, and (D) À300 mmHg for 10 min) were tested in 4 different days and each protocol was separated between 2 and 4 days. The selection of a range between 2 and 4 days was to accommodate the participant's schedule. The minimum of 2 days was selected to minimize the carry over effect of cupping therapy.

| DATA ANALYSIS
The two-way analysis of variance (ANOVA) with repeated measures was used to examine the main effect of pressure and duration factors and the interaction effect between the pressure and duration factors. The test of sphericity was used to examine whether assumptions were violated, including normal distribution. For post-hoc comparisons, Bonferroni correction was used. The dependent variables were oxyhemoglobin (Δ HbO 2 ½ in μM), deoxy-hemoglobin (Δ Hb ½ in μM), blood volume (Δ HbO 2 ½ þΔ Hb ½ in μM), and oxygenation (Δ HbO 2 ½ ÀΔ Hb ½ in μM). The independent variables were pressure (À225 and À300 mmHg) and duration (5 and 10 min). The significance level was set at p < 0.05. Statistical tests were implemented using SPSS 29 (IBM, Armonk, NY).   Table 2. The test of sphericity indicates that oxyhemoglobin data were normally distributed. Cupping therapy at À300 mmHg for 10 min results in the largest increase in oxyhemoglobin (6.75 ± 2.08 μM), and is significantly different from other three combinations, including at À225 mmHg for 5 min (2.58 ± 0.98 μM, p < 0.05), À225 mmHg for 10 min (3.42 ± 1.16 μM, p < 0.05), and À 300 mmHg for 5 min (3.65 ± 1.57 μM, p < 0.05) in Figure 2.
For deoxy-hemoglobin, the two-way repeated measures ANOVA shows that there is an interaction between the pressure and duration factors (F = 4.674, p = 0.045, and effect size = 0.216), and there is no main effect of the pressure factor (F = 0.716 and p = 0.409) and the duration factor (F = 0.034 and p = 0.855) in Table 2. The test of sphericity indicates that deoxy-hemoglobin data were normally distributed. Cupping therapy at À300 mmHg for 10 min (1.71 ± 0.78 μM) and À 225 mmHg for 5 min (1.22 ± 0.61 μM) results in a significant increase in deoxy-hemoglobin compared to the pre-cupping value, respectively ( p < 0.01) in Figure 3.
For blood volume, the two-way repeated measures ANOVA shows that there is an interaction between the pressure and duration factors (F = 4.921, p = 0.040, and effect size = 0.224), and there is a main effect of the pressure factor (F = 6.450, p = 0.021, and effect size = 0.275)   and the duration factor (F = 9.737, p = 0.006, and effect size = 0.364) in Table 2. The test of sphericity indicates blood volume data were normally distributed. Cupping therapy at À300 mmHg for 10 min results in a significant increase in blood volume (8.46 ± 2.43 μM) compared to À225 mmHg for 5 min (3.79 ± 1.35 μM, p < 0.05), À225 mmHg for 10 min (3.54 ± 1.53 μM, p < 0.05), and À300 mmHg for 5 min (4.10 ± 2.39 μM, p < 0.05) in Figure 4.

| DISCUSSION
This study provides evidence showing the main effect of the pressure and duration factors of cupping therapy and their interaction on the hemodynamic responses of the biceps muscle, including oxyhemoglobin, deoxy-hemoglobin, blood volume and oxygenation. Our results demonstrated that there is a significant interaction effect between pressure and duration factors on muscle deoxy-hemoglobin and blood volume, but not significantly on oxyhemoglobin and oxygenation. Our results also demonstrated that there is a significant main effect of cupping pressure on oxyhemoglobin, blood volume and oxygenation as well as a significant main effect of cupping duration on oxyhemoglobin, blood volume and oxygenation. Our findings are significant because different combinations of pressure and duration values cause different hemodynamic responses of the biceps muscle that may affect efficacy and effectiveness of cupping therapy on managing musculoskeletal impairment. This may partly explain the low evidence level of cupping therapy in the literature because most studies did not control the applied pressure and duration of cupping therapy. An insufficient combination of pressure and duration of cupping therapy may not improve muscle blood volume and oxygenation.
Recent studies suggest that muscle blood volume increases after cupping therapy [14]. However, it is largely unknown what intensities (in terms of pressure and duration) of cupping therapy can effectively induce a hyperemic response to benefit tissue for improving blood volume and oxygenation. In the present study, four combinations of cupping therapy including two durations (5 and 10 min) and two pressures (À225 and À300 mmHg) resulted in different hemodynamic responses of biceps muscle. Our results indicate that all four commonly used combinations of cupping therapy can significantly improve oxyhemoglobin, blood volume and oxygenation. This may partly contribute to the popular use of cupping therapy in athletes for improving muscle blood flow (e.g., increased oxyhemoglobin and oxygenation). Among four cupping protocols, cupping at À300 mmHg for 10 min induced a larger hyperemic response (blood volume) and metabolism (reflected as deoxy-hemoglobin) of the biceps. The negative pressure induced by cupping therapy results in a perpendicular force on the biceps muscle; and a larger perpendicular force can cause higher transmural pressure for inducing a larger hyperemic response after cupping therapy [10,17,26]. The transmural pressure is defined as the difference between the intramural blood pressure and the extramural blood pressure [27]. This may partly explain the results observed in this study.
The cupping pressure decides the magnitude of the perpendicular force applied on the muscle and may be the primary factor mediating muscle hemodynamic responses after cupping therapy [2,5,6]. One mechanism used to explain reactive hyperemia is related to the mechanical stimulus of the musculature during passive tension (area inside of cup) and compression (area under the rim of cup) which would then create a large hydrostatic pressure gradient between the microvasculature of the skin and muscle and its upstream conduit artery. The relaxation of vascular smooth muscle is a part of the myogenic response, which can change the transmural pressure. The vascular smooth muscle surrounding the blood vessel contracts under elevated pressure and relaxes under reduced pressure [28]. After the stimulus is moved, the vascular smooth muscle relaxes to dilate the blood vessel. Thus, the pressure of cupping therapy applied on the muscle needs to be sufficient to cause reactive hyperemia after cupping therapy. When the duration is longer, the effect of accumulated mechanical stress will be larger, which may induce a larger reactive hyperemic response. The magnitude of reactive hyperemia induced by different occlusion periods (e.g., standard 5 min and others 4, 6, and 8 min) determines the level of reactive hyperemia [29]. However, the specific effects of duration of cupping therapy on muscle hemodynamic responses require more studies.
An increased blood volume is mainly contributed by more oxyhemoglobin entering the cupping area after cupping therapy. Our results indicate that cupping therapy improved oxygen delivery in this study. The value of deoxy-hemoglobin (Δ Hb ½ ) can be used to assess the oxygen extraction capacity of the skeletal muscle. Lower deoxy-hemoglobin values as a marker for slower oxygen extraction have been reported in patients with mitochondrial myopathy. Their research demonstrated that a reduced oxygen extraction due to a decreased oxidative capacity in the skeletal muscle [30]. The deoxyhemoglobin response as a function of work rate has been observed during incremental cycle exercise [31]. In this study, cupping therapy at À300 mmHg for 10 min resulted in a largest increase in deoxy-hemoglobin that may indicate a higher metabolic rate under cupping therapy.
Oxygenation represents the balance between oxygen delivery and uptake, and cannot not distinguish between the two. The increased value of Δ HbO 2 ½ and the increased value of Δ Hb ½ of the biceps muscle in response to cupping therapy demonstrated that cupping therapy can improve the oxygen delivery and oxygen utilization of the muscle. Our findings further demonstrate that both pressure and duration factors affect muscle blood volume and oxygenation. Cupping therapy has potential to become a rehabilitation intervention to improve oxygenation for reducing muscle fatigue and improving muscle healing.
Previous studies investigated skin blood flow in response to cupping therapy using laser Doppler flowmetry [5]. In this study, NIRS was used to measure the oxygen delivery and oxygen utilization in the microvasculature of the skeletal muscle. The oxyhemoglobin has been selected as a measure of wash-in kinetics of oxygen during reactive hyperemia in the literature. After cupping therapy, an increase in oxyhemoglobin was observed in all four cupping protocols. The elevated oxyhemoglobin suggests that more oxygen after cupping therapy. These results support the benefits of using cupping therapy on improving blood flow (i.e., blood volume and oxygenation) to the muscle. Compared with pre-cupping value, the changed value of deoxy-hemoglobin is relatively small compared to oxyhemoglobin. Our results further demonstrate that a significant interaction effect between pressure and duration on deoxy-hemoglobin but not significantly in oxyhemoglobin. Although our study design using a 2 Â 2 factorial design could be used to assess the influence of pressure and duration of cupping therapy and their interaction on muscle blood volume and oxygenation, our study design in healthy participants does not allows the investigation of potential mechanisms for this difference between oxyhemoglobin and deoxy-hemoglobin.
Different combinations of pressures and durations of cupping therapy may cause different hemodynamic responses of the skin and muscle [2,5,6]. These studies demonstrated that the skin blood flow response was dependent on the applied pressure of cupping therapy [5]. The cutaneous microcirculation has similar mechanosensitivity as the microcirculation of the muscle to transmural blood pressure changes induced by cupping therapy. However, the skin is relatively incompressible compared to the muscle, and may not significantly respond to cupping therapy [32]. The hyperemic response of biceps muscle after cupping therapy exhibits a similar response pattern to skin blood flow [5,33]. A rapid increase in skin blood flow was observed after cupping therapy. The hyperemic responses after cupping therapy were significantly higher because all four combinations of cupping therapy were sufficiently to induce regulation of muscle blood flow. However, the same intensity (in terms of the combination of pressure and duration of cupping therapy) of cupping therapy may not induce a hyperemic response of the skin [5,33,34]. Thus, this study explored the magnitude of muscle hemodynamic response to cupping therapy at the same intensities used in previous research [5]. In this study, we further assessed blood volume in addition to oxyhemoglobin, deoxy-hemoglobin and oxygenation to comprehensively document microvascular responses of muscle to cupping therapy. These microvascular parameters could help determine effective dose response relationships of cupping therapy.
NIRS measurements usually use wavelengths in the range of 700-850 nm that is able to penetrate deeper in biological tissues such as the skeletal muscle. This region of wavelength can absorb chromophores such as oxyhemoglobin in micromolar concentration and deoxyhemoglobin in micromolar concentration. Unlike visible light (400-650 nm), the shorter wavelengths are unable to penetrate into tissues. Continuous wave (CW) is the broadest category of NIRS oximeters, which have been used to measure muscle oxygenation [35]. CW means that only changes in the light intensity are measured. This method allows the continuous quantification of relative values of venous oxygen saturation. In this research, CW NIRS was chosen to evaluate the response of muscle blood flow to different pressures and durations of cupping therapy. This study provides evidence that by using CW NIRS, researchers are able to evaluate the doseresponse relationship of cupping therapy on muscle blood flow. Future research can combine electromyography with NIRS to assess the change of muscle activity after cupping therapy to better understand the effect of cupping therapy.
This study provides the first evidence showing the effect of pressures and durations of cupping therapy on muscle blood volume and oxygenation as well as the interaction effect of pressure and duration on muscle blood flow. Significant hyperemic responses were observed after 4 cupping protocols in this study; and some combinations of pressure and duration of cupping therapy resulted in a significant higher increase in muscle blood flow, especially oxyhemoglobin and blood volume. NIRS was used in this study to document muscle blood flow changes after various intensities of cupping therapy for the first time. Our research methods using a 2 Â 2 factorial design could be used as a foundation to further investigate the effects of various pressure and duration values on muscle hemodynamic responses including oxyhemoglobin, deoxy-hemoglobin, blood volume and oxygenation and the dose-response relationship of cupping therapy. Based on our findings, cupping therapy could be an effective intervention on improving oxyhemoglobin, blood volume and oxygenation under sufficient intensities. Our findings also indicate a significant need to examine the skills of clinicians who practice cupping therapy by assessing the magnitude of negative pressure through applying fire cupping or manual suction device without pressure monitoring. Without the needed negative pressure, cupping therapy may not be able to induce changes of muscle hemodynamic responses for managing musculoskeletal impairment.
There are limitations of this study. First, this study was conducted in a homogenous group of participants. The results may not be generalized to people who have very different muscle thickness or body mass index. Future studies may need to consider these cofounding variables. Second, this research only studied the acute response of muscle blood volume and oxygenation after cupping therapy. It is unclear whether these different combinations of cupping therapy could affect long-term effect of cupping therapy on improving muscle blood flow. Future research may need to investigate the longterm effect of cupping therapy. Third, this study used an fNIRS device to measure hemodynamic responses of the muscle. Up to date, there is no guideline on the use of fNIRS on measuring muscle blood volume and oxygenation. In this study, we followed the standard operations of an fNIRS device. Fourth, we adopted a counterbalanced design to minimize the carry over effect of various cupping therapy protocols. We have recruited only 18 participants that is not sufficient for a full rotation of 24 participants. Last, there were more females than males in this study. It is unclear whether the gender effect would significantly affect muscle hemodynamic response to cupping therapy. Future studies should use a gender balanced subject population.

| CONCLUSIONS
In this study, we demonstrate that the main effect of pressure and duration of cupping therapy and their interaction on muscle blood flow. To the best of our knowledge, this is the first study investigating the effect of various intensities of cupping therapy on muscle blood volume and oxygenation. An appropriate combination of pressure and duration of cupping therapy should be determined for improving efficacy of cupping therapy on managing musculoskeletal impairment.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.