Meridian energy analysis of the immediate effect of coffee consumption

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ScienceDirect European Journal of Integrative Medicine 6 (2014) 74–81

Original article

Meridian energy analysis of the immediate effect of coffee consumption Ming-Yen Tsai a,b , Chun-En Kuo a , Yu-Chuen Huang b,c , Ching-Liang Hsieh b,c , Yung-Hsiang Chen b,c,∗ , Wen-Chi Chen b,c,∗ a

Graduate Institute of Integrated Medicine, School of Chinese Medicine, College of Chinese Medicine, Acupuncture Research Center, China Medical University, Taichung 40402, Taiwan b Department of Traditional Chinese Medicine, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung 83301, Taiwan c Departments of Chinese Medicine, Urology, and Medical Research, Genetics Centre, China Medical University Hospital, Taichung 40402, Taiwan Received 20 March 2013; received in revised form 26 August 2013; accepted 4 September 2013

Abstract Introduction: Coffee consumption has immediate physiological, behavioral, and subjective effects. Despite its widespread use, few studies have described the impact of energy distribution and transformation in meridian vessels. The purpose of this study was to use a Ryodoraku instrument to measure the changes of meridian electrical conductance in healthy volunteers before and after coffee consumption. Methods: In thirty-one healthy volunteers, the meridian electrical conductance, blood pressure (BP), heart rate (HR), and index of sympathovagal balance were continuously recorded before and after drinking a cup of instant coffee. Results: Statistical analysis showed that 30 min after coffee consumption, the mean values of electrical conductance increased on most meridians except the bilateral Triple Energizer, Liver, and Stomach, as well as the left Kidney and Large Intestine. From 30 to 60 and 60 to 120 min, the electrical conductances were maintained significantly only on the bilateral Spleen, Urinary Bladder, and left Pericardium meridians, as well as on the bilateral Spleen, Urinary Bladder, and right Kidney meridians, respectively. The average index of sympathovagal balance decreased significantly at 30 min, while BP increased and HR decreased. Conclusion: These findings suggest changes in the specific energy distribution and transformation in the meridian vessels after coffee consumption. Coffee consumption has a similar immediate effect on the cardiovascular system, including increase in BP and decrease in HR. Interestingly, coffee has a temporary stabilizing effect on the higher index of sympathovagal balance. This response may explain why the index is not relevant to the autonomic nervous system but is relevant to mental tasks after coffee consumption. © 2013 Elsevier GmbH. All rights reserved. Keywords: Coffee; Caffeine; Ryodoraku; Meridian; Electrical Conductance; Autonomic nervous system

Introduction Coffee, has been brewed from roasted coffee beans for almost 700 years and is the most popularly consumed beverage worldwide, second only to water, with a worldwide consumption of approximately 10 million tons per year [1,2]. Owing to the high consumption of coffee, it has been investigated ∗

Corresponding authors at: Graduate Institute of Integrated Medicine, China Medical University, No. 91, Hsueh-Shih Road, Taichung 40402, Taiwan. Tel.: +886 4 22053366x3512; fax: +886 4 22037690. E-mail addresses: [email protected], [email protected] (Y.-H. Chen), [email protected] (W.-C. Chen). 1876-3820/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.eujim.2013.09.002

extensively to determine the either beneficial or detrimental effects on the human organism [3–5]. Studies have shown that coffee consumption can promote neuroprotective, antioxidant, and metabolic activities [6–8]. However, some controversy regarding its risks still exists, due to the association of coffee consumption with an increased risk of developing cardiovascular complications and cancer [9,10]. In fact, coffee is a complex chemical mixture containing more than a thousand compounds [11]. At intake levels associated with coffee consumption, caffeine appears to exert most of its biological effects through stimulating sympathetic and psychoactive systems, resulting in central nervous system stimulation, increased blood pressure, increased metabolic rate, sleep disorder, and diuresis [12].

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According to Traditional Chinese Medicine (TCM) and “Qiblood” theory, all the stimulation and ingestion is seen to flow into and affect specific cells, tissues, and organs through different meridians [13]. It is generally assumed that the electrical conductivity measurements of meridians provide information about the balance of bioenergy or Qi-blood in the body [14,15]. The Ryodoraku mechanism for measuring electrical activity of meridians, was developed by Dr. Yoshio Nakatani, in the 1950s, reflects the condition of certain organs by analyzing and comparing their mutual relations and according to changes in micro-electrical currents [16]. The electrical conductance of the acupuncture points of human subject can be measured by a computerized testing instrument with a very low electrical current. The Source (Yuan) acupoints on the wrists and ankles, (the places where the Yuan Qi of the corresponding Zang-Fu organs passesthrough abundantly), have the property to represent the relevant meridian [17,18]. Several studies have used this mechanism to investigate changes in human moods or the mechanism of the sympathetic system [19,20]. Moreover, Ryodoraku theory has been shown to be a supplementary diagnostic method for selective diseases and a useful parameter for evaluating therapeutic effects of acupuncture [21,22]. However, there has been no previous no research concerning the application of Ryodoraku theory for the detection of the diverse effects of coffee intake and chronic coffee consumption. In the present study, we used a device based on the electrical conductance theory to measure the association of sympathetic nerves and meridian energy on instant coffee consumption in normal human participants. The purpose of this study was to evaluate whether measurement of the electrical conductance of healthy people who consumed coffee could be used to predict meridian trends and potential health effects. Materials and methods Sampling method Thirty-one healthy volunteers (15 men and 16 women) aged 25–45 years [29.1 ± 4.5 years, mean ± standard deviation (S.D.)] participated in the study. Most of the participants (29/31) consumed <1 cup of coffee per day. The mean duration of coffee consumption was 2.1 years. All participants worked in a hospital. The average body-mass index (BMI) value was 21.7 ± 3.6 kg/m2 . The reasons for exclusion were as follows: (1) women menstruating or pregnant, (2)currently smoking, drinking and/or chewing betel nut, and (3) currently taking pharmaceuticals or dietary supplements. All participants were asked to avoid intake of either caffeinated beverages or diets including “coffee, wine, chocolate, and tea” in the 3 days preceding and during the experiments. The recruitment of participants and the study design were approved in case No. 100-4025A3 of the Chang Gung Institutional Research Board. Coffee preparation A soluble sample of 2.3-g instant coffee was prepared in the standard amount (200 ml) by dilution in hot water. The instant

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Table 1 The labeled and true amount of caffeine in the coffee package.

Caffeine (mg/g) Protein (%) Special gravity

Label content

SGS result

56.5 21.7 N/A

46.8 22.1 1.007

coffee was the brand Starbucks VIA® Ready Brew Italian Roast (The Zuoying THSR store, Kaohsiung, Taiwan). As shown in Table 1, the caffeine content on the label of one coffee package was 56.5 mg caffeine/g (5.65%). However, the true content of caffeine was 4.68%, as analyzed by SGS Taiwan LTD. The coffee was brewed at 85–95 ◦ C and served at 50–60 ◦ C, and the participants were asked to drink the entire volume within 10 min under supervision. No additives (such as milk or sugar) were allowed. All participants were instructed not to consume anything else except mineral water during the experiments. Study design and procedure The electrical conductance of an acupoint was measured using a Meridian Energy Analysis Device (MEAD100, MedPex Enterprises, Taichung, Taiwan) operating at DC 12 V and 0 to 200 ␮A. The method and procedure of the MEAD is based on the Ryodoraku theory and is similar to equipment used in previous studies [15,16]. Higher values indicate higher conductance between the reference electrodes. One metal cylinder is held in the right hand of the participant. The other, a springloaded probe, is moistened with 5% saline and, after sterilization, applied sequentially to the 24 Source (Yuan) acupoints located along the 12 meridians: Lung (Taiyuan, LU9), Pericardium (Daling, PC7), Heart (Shenmen, HT7), Small Intestine (Wangu, SI4), Triple Energizer (Yangchi, TE4), Large Intestine (Hegu, LI5), Spleen (Taibai, SP3), Liver (Taichong, LR3), Kidney (Taixi, KI3), Urinary Bladder (Jinggu, BL65), Gallbladder (Qiuxu, GB40), and Stomach (Chongyang, ST42) (Fig. 1). After 2.5 s the device was automatically interrupted, and the average value was recorded on a computerized system. The environmental condition was controlled by maintaining a room temperature of 26 ◦ C and 28 ◦ C, and moisture was kept constant. The participants were asked to lie flat and rest for 15 min in a stable mood. All measurements are performed by one operator. They were first measured between 7:00 am and 9:00 am, 1 h after consuming a light breakfast. The first measurement, taken before instant coffee ingestion, was used as the baseline values of acupoint conductance (designated as point X). After participants ingested a cup (200 ml) of instant coffee and rested for 30 min, second measurements were taken. Subsequent measurements of the acupoint conductances followed at 30, 60, and 120 min (designated as points A, B, and C, respectively). Also recorded was the index of sympathovagal balance, defined as the ratio of the highest and the lowest average values of electroconductivity on the dorsal or ventral side during the MEAD analysis. To confirm the stability of the MEAD data, we followed the model of Huang et al. [23]. Before the study was conducted, 31

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Fig. 2. Experimental procedure. I, ingesting instant coffee; R, acupoint conductance; X, baseline measurement; A, 30-min measurement; B, 60-min measurement; C, 120-min measurement.

of the 24 meridians at baseline, 30 min, 60 min, and 120 min after instant coffee consumption were calculated. In order to highlight the differences between individual subjects, the variations in the significant meridians’ conductance in each time segment (time between A–X, B–X, and C–X) were converted to percentage differences [%D(Ti) = R(Ti)-R(T0)/R(T0) × 100%]. Where R is the Ryodoraku value, Ti is the observation time relative to instant coffee ingestion (time at point A, B, and C), and T0 is the baseline time (time at point X). All cardiovascular measurements during the period were taken as the subject sat semi-recumbent in a recliner chair. Blood pressure (BP) was measured with a mercury sphygmomanometer (SM-100, Yagiden, Japan). Results are reported as mean ± S.D. Data were analyzed by paired samples t-test. A value of P < 0.05 was considered statistically significant. Fig. 1. The schematic Ryodoraku diagram and 24 detected acupoints based on traditional Chinese meridian theory. (A) The experimental setup; (B) The detected acupoints symmetrically located on each limb. (LU9, Lung; PC7, Pericardium; HT7, Heart; SI4, Small Intestine; TE4, Triple Energizer; LI5, Large Intestine; SP3, Spleen; LR3, Liver; KI3, Kidney; BL65, Urinary Bladder; GB40, Gallbladder; ST42, Stomach.)

participants were recruited to examine the MEAD measurements twice for everyone, 15 min apart. The correlation of the two measurements in the same subject was high (r = 0.85), suggesting that the MEAD data were reliable. The findings of physical effect after coffee ingestion, included (1) psychological symptoms (anxiety, restless, excitement, fatigue, and alertness), (2) cardiovascular symptoms (palpitation, flush, shortness of breath, and chest tightness), (3) neurological symptoms (dizziness, headache, and tremor), (4) gastrointestinal symptoms (nausea, vomiting, diarrhea, and abdominal pain), and (5) other symptoms (thirst and frequency of urination) which were recorded “yes” or “no” at every Ryodoraku measurement. Blood pressure and heart rate were simultaneously measured throughout the study. The measurement schedule is shown in Fig. 2. Statistical analyses Data were entered into and analyzed with SPSS version 18.0 (SPSS, Inc). For each subject, the average conductance values

Results The 12 meridian vessels have 24 acupoints on the left and right sides. The conductances of the 24 acupoints measured with mean values are listed in Table 2. We first analyzed the overall electrical conductance in different meridians before coffee drinking (Fig. 3). At 30 min after coffee drinking, the mean value of electrical conductance significantly increased on the bilateral Lung, Pericardium, Heart, Small Intestine, Spleen, Urinary Bladder, and Gallbladder meridians, as well as on the right Large Intestine and Kidney meridians (P < 0.05). At 60 min, the mean value of electrical conductance significantly increased on the bilateral Spleen and Urinary Bladder meridians, as well as on the left Pericardium meridian (P < 0.05). At 120 min, it increased significantly only on the bilateral Spleen and Urinary Bladder meridians, as well as the right Kidney meridian (P < 0.05). The results of percentage difference of significant meridians’ conductance are shown in Fig. 4. As shown in Table 3, the index of sympathovagal balance was 14.88% lower than baseline at 30 min after coffee drinking (P = 0.02). BP was significantly elevated at 30 min after coffee drinking (P = 0.02). Accordingly, mean heart rate was 2.36 bpm lower than baseline at 30 min (P = 0.02), and 5.09 bpm lower than baseline at 60 min (P < 0.01). The frequencies of physical effect were reported after coffee consumption (Fig. 5). Psychological symptoms were found in

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Table 2 Mean values and (S.D.) of the 24 acupoint conductances in coffee consumption (N = 31). LLU

RLU

LPC

RPC

LHT

RHT

LTE

RTE

LSI

RSI

LLI

RLI

X

66 (30)

64 (27)

62 (28)

59 (26)

52 (27)

50 (24)

54 (33)

49 (26)

55 (32)

56 (32)

57 (30)

52 (27)

A

76 (23)

76 (24)

71 (21)

68 (26)

61 (21)

57 (26)

61 (25)

56 (25)

71 (27)

67 (30)

67 (24)

65 (26)

B

72 (22)

71 (23)

71 (21)

64 (23)

59 (22)

53 (24)

50 (22)

46 (23)

65 (24)

59 (29)

62 (22)

53 (21)

C

66 (25)

69 (24)

66 (23)

62 (24)

55 (22)

51 (22)

48 (24)

45 (22)

60 (30)

56 (28)

49 (21)

50 (22)

LSP

RSP

LLR

RLR

LKI

RKI

LST

RST

LBL

RBL

LGB

RGB

X

54 (25)

50 (23)

49 (27)

49 (27)

47 (28)

41 (25)

40 (24)

39 (23)

48 (20)

44 (20)

31 (22)

35 (26)

A

63 (23)

58 (21)

55 (27)

56 (27)

52 (24)

49 (24)

46 (24)

43 (22)

55 (18)

52 (18)

38 (23)

41 (28)

B

62 (23)

58 (22)

53 (27)

54 (28)

53 (23)

49 (24)

44 (23)

42 (21)

57 (19)

54 (18)

36 (23)

37 (22)

C

61 (23)

59 (22)

46 (27)

45 (27)

51 (25)

48 (23)

40 (25)

37 (22)

58 (21)

53 (19)

32 (22)

33 (24)

Data are shown as the mean (S.D.) values. L, left; R, right; LU, lung; PC, pericardium; HT, heart; TE, triple energizer; SI, small intestine; LI, large intestine; SP, spleen; LR, liver; KI, kidney; ST, stomach; BL, urinary bladder; GB, gallbladder; X, baseline measurement; (A) 30-min measurement; (B) 60-min measurement; (C) 120-min measurement.

Fig. 3. Bar graph indicating the 24 acupoint electrical conductances after coffee drinking, achieved at baseline, 30, 60, and 120 min after consumption. (*P < 0.05, statistically significant difference from baseline values.) Table 3 Effect of a single cup of the instant coffee consumption on the index of sympathovagal balance, SBP, DBP and HR. Baseline Blood pressure Systolic BP (mmHg) Diastolic BP (mmHg) Heart Rate (bpm) Sympathovagal Balance Index (normal: 1.0–1.5) Values represent as the mean ± S.D. * P < 0.05, statistically significant from baseline value.

113.8 72.8 68.4 2.2

± ± ± ±

30 min 15.1 11.8 9.4 0.8

119.3 77.7 66.1 1.8

± ± ± ±

60 min 16.9* 8.6* 8.4* 0.6*

113.1 74.6 63.2 2.1

± ± ± ±

120 min 16.8 10.1 7.1* 0.9

117.2 73.2 67.5 2.2

± ± ± ±

16.2 12.1 9.4 0.8

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Fig. 4. The percentage differences in electrical conductance for significant meridians (during the three intervals AX, BX, and CX). L, left; R, right; LU, Lung; PC, Pericardium; HT, Heart; SI, Small Intestine; LI, Large Intestine; SP, Spleen; KI, Kidney; BL, urinary Bladder; GB, Gallbladder.

Fig. 5. The physical effects after acute coffee consumption at baseline, 30, 60, and 120 min.

48.4, 38.7, and 19.4% of the participants at 30, 60 and 120 min, respectively. Sequentially, cardiovascular symptoms were found in 16.1, 22.6, and 9.7% of participants; neurological symptoms were found in 16.1, 16.1, and 9.7%; and gastrointestinal symptoms were found in 16.1, 22.6, and 6.5%. Accordingly, other symptoms including thirst and frequency of urination were found with in 35.5, 25.8, and 16.1%. Discussion Coffee is a widely popular beverage in Taiwan, but the habits and ethnic makeup of coffee drinkers in Taiwan differ from those in western countries. In 2010, the average consumer consumed more than 78 cups/year (200 ml/cup; 100–200 mg caffeine). Taiwanese statistics indicate that 44% of adults consume coffee on a daily basis, while only 2% are heavy drinkers (more than 3 cups/day). Nearly all volunteers in our study were non-habitual coffee drinkers; therefore, their tolerance to coffee and occurrence of withdrawal symptoms should be quite low [24,25]. In addition, we found a 17.2% difference in the caffeine content between a labeled package and the content listed in the SGS report. This result indicated that the caffeine content listed on product labels on the market could be inaccurate, although the difference may have been the result of measurement error or differences in product batches. According to one report, the average rate of consumption of caffeinated coffee in Taiwan is still within the current daily guidelines [26].

TCM theory indicates that each food or drug follows its own metabolic process through the flow of “Qi” and “blood” in meridians, and then the energy is transferred into the specific inner organs. A previous study has shown that changes in the energy distribution of the meridian system can be measured by the Ryodoraku device after immediate glucose ingestion in different physical conditions [27]. Our measurements showed that the energy distribution on meridians changed and symptoms of stimulation presented after coffee consumption. Coffee ingestion has previously been associated with an increase in skin conductance [28,29], suggestive of the changes in electrodermal activity and an increase in Ryodoraku values [30]. Actually, the increase in electrical conductance was not found on all meridians, but only on specific ones, and decreased over time. The follow-up measurement showed, as expected, that the effect of coffee consumption on the meridian system is reliable and verifiable. Caffeine appears to exert most of its biological effects at the intake of coffee. Caffeine is absorbed from the gastrointestinal tract into the blood stream within 20 min, its peak plasma concentration is reached within 40–60 min, and its halflife is 2.5–4.5 h [31]. Once absorbed, caffeine exerts a variety of pharmacological actions at diverse sites, both centrally and peripherally [12,32]. The results of this study revealed the energy distribution on the meridian system after coffee consumption and might provide additional information for current caffeine pharmacology [33,34]. Interestingly, the significant increase in electrical conductance on the Triple Energizer, Stomach, and Liver meridians was entirely absent, which merits further study. According to TCM, the Stomach only a storage organ to receives food and drink, while the Spleen governs the transportation and transformation of energy after digestion. The Spleen is also related to antioxidant defense systems against free radical damage and protective effect on the digestive tract [35]. Thus, it appears that the activation of the Spleen meridian within 2 h after coffee consumption is the key factor in coffee accelerating digestion, enhancing antioxidation, and increasing the metabolic rate [9,36,37]. Additionally, the Liver and Triple Energizer meridians can govern conveyance and dispersion to regulate and distribute Qi and interstitial fluid, respectively [13,38]. Although the caffeine in coffee is metabolized in the liver by cytochrome P450 enzymes and has been found to be correlated with a decreased risk of chronic liver disease [39,40], it is not clear, from our data, why the supplied energy is not available on the Liver meridian after coffee consumption. This unavailability is probably due to the meridians not being parallel to the current anatomy and physiology of the organ [41]. In addition, the energy on the Kidney and Urinary Bladder meridians could even last ≥2 h. According to TCM, the Kidney is storing will and is a complement to Urinary Bladder each other. The fluctuation of Kidney “Qi” can strengthen neuroprotective function, furthermore, affect the Urinary Bladder’s function, causing changes in the amount, time, and frequency of urine output [42]. This supports the spatial learning, memory, and diuretic effects associated with coffee consumption [6,32]. The study results show that some differences were found between right and left side of the meridian after consuming coffee. There may be relevant to diurnal cycle

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of Qi-blood in meridians or balance of yin and yan derived from immediate coffee consumption [43]. For example, the energy promotion after consuming coffee is presented mainly on Kidney “Qi” and Large Intestine “Qi” in the morning. This perhaps is why bilateral Kidney and Large Intestine are not matched. Distinct elevation in the electrical conductances on the Heart and Pericardium meridians can be transiently observed after coffee consumption. In TCM theory, both meridians represent the cardiovascular system and some central functions of the brain. Recent studies have shown that consumption of coffee is associated with improvements in recognition, attention, and short-term memory [5,44,45]. Our data also confirm previous reports of a presser effect, followed by baroreceptor-mediated inhibition of heart rate, of acute coffee drinking in humans. The presser effect of coffee has been well studied, and the mechanisms appear to be related to activation of the central or peripheral sympathetic nervous system [46–48]. Many previous studies have shown that the autonomic (sympathetic) nervous system is somehow related to the meridians [14,49]. A study of Ryodoraku application on renal colic patients suggested that the index of sympathovagal balance may be used as a valuable diagnostic method for sympathetic activity in the body [22]. However, the coffee drinkers in this study did not show a synchronous effect in the index of sympathovagal balance while BP was increasing. This result reveals that the change in the meridians’ index cannot fully be explained by the autonomic nervous system, and that brain activity must be taken into consideration. It is worth noting that coffee consumption alleviated the higher sympathovagal index for most of the participants in this study. Ryodoraku theory indicated that the hospital workers were in a tense and tired physical condition if the index was in disequilibrium. Some reports have suggested that coffee consumption can improve mood, including increased happiness, a reduction in depressive symptoms, and decreased anxiety [50]. This result might have potentially interesting implications of a mood-modulating effect of coffee in an everyday setting. Other physiological effects associated with immediate coffee consumption include bulbar breath center stimulation, promote digestion, and increased colonic motility [51,52]. These also are in accordance with TCM consideration that the Lung controls breathing and Small Intestine/Large Intestine regulate nutrients absorption and transportation. However, the energy distribution and transformation in various meridians at different times might be contributed to additional compounds in coffee that could have metabolic importance are present in small concentrations, including cafestol, kahweol, chlorogenic acid, and micronutrients [9]. Coffee contains hundreds of compound such as caffeine, carbohydrates, lipids, nitrogenous compounds, vitamins, minerals, alkaloids and phenolic compounds. Their biological, metabolism, and pharmacokinetic activities might affect different meridians and energy distribution. The goal of our study was to describe the continuous phenomenon changes of meridian electroconductivity before and after coffee ingestion. The baseline value before coffee consumption was served as control. However, this study is exploratory and cannot provide definitive data because the lack on a placebo (with warm water). This study also has other

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limitations. Firstly, as there were no previous studies to rely on, the comparisons of meridian conductance for heavy coffee drinkers, different products of coffee, decaffeinated coffee, or other caffeinated beverages are not fully understood. Secondly, coffee is a readily available and commonly ingested source of caffeine. However, coffee also contains hundreds of other biologically active compounds, many of which are metabolically distinct from caffeine [53]. Our study could not rule out the potential pharmacological effects of these other compounds. Third, the effects of light breakfast and coffee on the meridian were difficult to distinguish because some degree of gastrointestinal upset was intolerable for volunteers during overnight fasting. Fourth, the measurement of acupoint conductance still exhibits considerable variations. Although we have tried to control experimental errors and using the correlation coefficient to confirm the stability of Ryodoraku values [54,55]. Further studies will take this factor into consideration. Conclusion The present study showed that, as determined by the Ryodoraku mechanism, some types of meridian electrical conductance were elevated after coffee consumption, and the energy was sustained for at least two hours. The results suggest that the immediate effects of coffee consumption may result in part from a conditioned response, from the viewpoint of TCM. We also noticed that the decrease in the index of sympathovagal balance was significant at 30 min after coffee drinking and then returned to baseline. In this study, however, the index of sympathovagal balance did not provide sufficient evidence for analysis of autonomic response. Conflict of interests The authors declare that there are no conflict of interest. Authors’ contributions M.Y. Tsai created the study design and coordination, conceived the study, and helped to draft the manuscript. C.E. Kuo performed subject recruitment and device operation. Y.C. Huang conducted all statistical analyses and interpretation of the data. Y.H. Chen, C.L. Hsieh, and W.C. Chen reviewed the manuscript and supervised during the study. All authors have read and approved the paper. Acknowledgments This study is supported in part by the Taiwan Department of Health Clinical Trial and Research Center of Excellence Grant (DOH102-TD-B-111-004) and the National Science Council (NSC 102-2320-B-039-025). We would like to thank ChihCheng Huang for his professional assistance in drawing the figures and tables. We also would like to thank all the participants who participated in this study for their valuable contribution.

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