Effects of regular high-cocoa chocolate intake on arterial stiffness and metabolic characteristics during exercise

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The effects of regular high-cocoa chocolate intake on arterial stiffness and metabolic characteristics during exercise Masato NISHIWAKI Ph.D. , Yuri NAKANO B.Sc. , Naoyuki MATSUMOTO Ph.D. PII: DOI: Reference:

S0899-9007(18)30941-9 https://doi.org/10.1016/j.nut.2018.09.021 NUT 10328

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Nutrition

Please cite this article as: Masato NISHIWAKI Ph.D. , Yuri NAKANO B.Sc. , Naoyuki MATSUMOTO Ph.D. , The effects of regular high-cocoa chocolate intake on arterial stiffness and metabolic characteristics during exercise, Nutrition (2018), doi: https://doi.org/10.1016/j.nut.2018.09.021

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HIGHLIGHT High-cocoa chocolate intake reduces arterial stiffness in youth

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Arterial stiffness is independently reduced after adjusting blood pressure

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High-cocoa chocolate intake does not affect exercise metabolic characteristics

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Regular cocoa chocolate intake is unlikely to reduce body mass and fat

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The effects of regular high-cocoa chocolate intake on arterial stiffness and

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metabolic characteristics during exercise

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High-cocoa chocolate intake and cardiometabolic health

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Ph.D.2

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Masato NISHIWAKI Ph.D.1, Yuri NAKANO B.Sc.1, and Naoyuki MATSUMOTO

Faculty of Engineering, Osaka Institute of Technology, Osaka, Japan

2)

Faculty of Environmental Symbiotic Sciences, Prefectural University of Kumamoto,

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1)

Kumamoto, Japan

Role of each author in the work: Conceived and designed the study: MN and YN. Performed the study: MN and YN. Analyzed the data: MN and YN. Wrote the paper: MN, YN, and NM. Interpreted the

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data: MN and NM. All authors approved the final version of the article.

Acknowledgments: We sincerely thank the study participants for their cooperation. The research did not

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receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

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Word count: 4982; Figures: 2, Tables 2, Supplementary material 2.

Corresponding author: Masato NISHIWAKI

Faculty of Engineering, Osaka Institute of Technology 5-16-1

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Ohmiya, Asahi-ku, Osaka 535-8585, Japan, Phone: (+81) 6-6954-4391, Fax: (+81) 6-6952-9976,

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E-mail: [email protected]

The Human Ethics Committee at the Osaka Institute of Technology approved the study

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(approval number: 2016-47), which proceeded according to the guidelines of the Declaration of Helsinki. The study was also registered in the UMIN Clinical Trials

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Registry (UMIN000026486).

High-cocoa chocolate intake and cardiometabolic health

ABSTRACT

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Objective: This study aimed to examine the effects of regular high-cocoa chocolate consumption on arterial stiffness and fat oxidation during light-to-moderate intensity exercise. Methods: This randomized, controlled, parallel-group intervention study included 32 Japanese college students (mean age, 20.7 ± 0.3 y; male, n = 24; female, n

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= 8) who were assigned to either control or intervention groups (n = 16 each). The control group did not alter their habitual diet or physical activity throughout the study period. The intervention group consumed a total of 20 g/day (508 mg of cacao

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polyphenol) of high-cocoa chocolate for four weeks. Blood pressure, heart-ankle pulse wave velocity (haPWV), cardio-ankle vascular index (CAVI), body composition, and metabolic characteristics during exercise at 50% maximal oxygen uptake level were assessed before and after the intervention. Results: Four weeks of high-cocoa chocolate

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ingestion significantly reduced haPWV and CAVI (%change, intervention vs. control: -2.3 ± 0.9% vs. 0.9 ± 0.9%, and -4.8 ± 1.8% vs. 0.7 ± 1.3%, respectively; both P < 0.05).

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However, blood pressure, weight, body mass index, body fat, waist circumference, and

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metabolic characteristics during exercise, such as glucose, lactate, respiratory exchange ratio and rates of substrate oxidation of carbohydrate and fat, did not significantly

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change in either group. Conclusions: Four weeks of regular high-cocoa chocolate consumption reduced arterial stiffness after considering BP in young healthy males and

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females. However, the habitual consumption of high-cocoa chocolate for four weeks did not affect metabolic characteristics during light-to-moderate intensity exercise and body composition. KEY WORDS: Arteriosclerosis, Blood pressure, Cardio-ankle vascular index, Fat oxidation, Light-to-moderate intensity, Pulse wave velocity INTRODUCTION

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Fruits, wines, and chocolate are polyphenol-rich, and their beneficial effects are thus attracting increasing attention [1-4]. Recent meta-analyses have shown that high-cocoa chocolate or cocoa products favorably affect cardiometabolic health [1-3]. Most individuals can easily intake chocolate, and thus high-cocoa chocolate could be an

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effective cardiovascular preventive strategy for large populations [5]. Therefore, further scientific evidence of the benefits of high-cocoa chocolate is needed.

Pulse wave velocity (PWV) generally serves as an index of arterial stiffness and

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increased arterial stiffness has been identified as an independent risk factor for future cardiovascular disease and mortality [6]. A prospective analysis has indicated that weekly chocolate intake benefits arterial stiffness [7]. Indeed, several previous studies have shown that arterial stiffness is reduced by cocoa products and that flow-mediated

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dilation (FMD) is increased by the habitual consumption of high-cocoa chocolate intake [2, 4, 8]. Aside from these studies, however, little is known about whether regular

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high-cocoa chocolate consumption directly affects arterial stiffness. Pereira et al. found

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that high-cocoa chocolate simultaneously reduced arterial stiffness, central BP, and brachial BP in young healthy individuals [9]. Many other studies have demonstrated

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that cocoa consumption reduces BP [2, 9, 10], but BP is a powerful confounding factor that affects PWV [11, 12]. Interventions with high-cocoa chocolate have also indicated

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that the magnitude of reduction in BP is associated with baseline BP value, and several studies, particularly those that included normotensive persons, found that BP did not improve regardless of intake dose [2, 10]. Accordingly, these findings raise the question of whether regular high-cocoa chocolate ingestion directly reduces arterial stiffness or reduces BP and consequently reduces arterial stiffness, especially in young healthy individuals. To the best of our knowledge, the effects of high-cocoa chocolate on

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arterial stiffness and issues regarding BP have not been comprehensively addressed. The habitual intake of high-cocoa chocolate/cocoa products confers beneficial effects on metabolism-related biomarkers [1, 3]. Acute high-cocoa chocolate ingestion can increase the mobilization of free fatty acids (FFA) or triglycerides (TG) during

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light-to-moderate intensity exercise [13, 14], suggesting that high-cocoa chocolate consumption has beneficial effects on exercise as part of weight-loss programs. In

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general, plasma FFA flux rapidly peaks at light-to-moderate exercise intensity of 45% ． 50% maximal oxygen uptake (VO2max), and lipids are thought to provide slightly more than half the energy consumed at rest and during light-to-moderate intensity exercise [15]. If regular high-cocoa chocolate consumption actually increases fat oxidation, changes in metabolic parameters could be determined by measuring respiratory gas

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exchange during exercise.

Based on this background, we tested the hypothesis that regular high-cocoa chocolate

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consumption induces a reduction in arterial stiffness and/or an increase in fat oxidation

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during light-to-moderate intensity exercise in young healthy individuals.

METHODS

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Participants

Thirty-two healthy Japanese college students (mean age, 20.7 ± 0.3 y; male, n = 24;

female, n = 8) at our institution participated in this randomized, controlled, parallel-group intervention study. None of them were under medication or had participated in a regular exercise program for at least one year. The participants were matched for age, sex, BP, and arterial stiffness as closely as possible and randomly

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assigned to either a control (n =16) or an intervention (n = 16) group (Fig. 1). The purpose, procedures, and risks of the study were explained to all of the students and they provided written informed consent before participating in the study. The Human Ethics Committee at the **** approved the study (approval number: *****), which

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proceeded according to the guidelines of the Declaration of Helsinki. The study was also registered in the UMIN Clinical Trials Registry (****).

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Sample size and experimental procedures

We determined the sample size for each group by power calculations using G*Power 3 and assumed that arterial stiffness would be reduced by approximately 5% according to previous findings [2, 9, 16]. To detect this difference at 80% power with a two-tailed

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of 5%, the intervention group should comprise 10 - 16 participants. Thus, we assigned 16 participants each to the control and intervention groups.

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Both groups were assessed before and after the study. All tests proceeded in a quiet

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air-conditioned room (22°C - 24°C) at the same time day and at the same number of hours after the last meal to avoid potential diurnal variations. The participants were

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required to abstain from caffeine and fast for ≥ 4 h before each test.

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Body composition

Weight, body fat, lean body mass (LBM), and body mass index (BMI) were

determined by bioelectrical impedance using a TBF-410 instrument (Tanita Co., Tokyo, Japan) as described in our previous study [12]. Waist circumference was measured around the abdomen at the level of the navel at the late expiratory phase using a tape measure. Day-to-day coefficients of variation (CV) for each parameter were all < 10%

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under our experimental conditions [12, 16, 17].

Arterial stiffness, blood pressure, and heart rate We measured PWV, blood pressure (BP), and heart rate (HR) with the participants in

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the supine position using a semi-automated device (VS-1500AE/AN; Fukuda Denshi, Tokyo, Japan) [12, 16, 17]. Heart-ankle PWV (haPWV) and the cardio-ankle vascular index (CAVI) were calculated as indicators of arterial stiffness [12, 16, 17]. The

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carotid-femoral PWV (cfPWV), which is an index of central arterial stiffness, was measured using the same device in the control (n = 15) and intervention (n = 16) groups [18]. The intraobserver CV for haPWV, CAVI, and cfPWV determined in the laboratory on two separate days were 2.6 ± 0.6%, 3.6 ± 0.6%, and 7.5 ± 1.2%, respectively [12,

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16-18].

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Metabolic parameters at rest and during submaximal exercise

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Rest and exercise proceeded on an electromagnetically braked cycling ergometer (75XL III; Konami, Tokyo, Japan) in the upright position. After a 5-min rest, 6 min of

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exercise proceeded at a constant submaximal intensity according to previous protocols of 45, 70, and 100 W for men and 20, 35, and 50 W for women. Pedaling rates remained

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constant at 60 rpm [19, 20]. ． Minute expired ventilation (VE), oxygen uptake (VO2), and carbon dioxide output ． (VCO2) were collected and determined every 15 s using an automatic gas analyzer with a mixing chamber (AR10; Arco System, Chiba, Japan). Heart rate was monitored using an HR monitor (V800; Polar Japan, Tokyo, Japan). The data were averaged during the last 2 min of the 5-min rest and the six-minute bout of exercise at each intensity.

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Respiratory exchange ratios (RER), rates of substrate oxidation of carbohydrate (CHO) and fat, and the relative contribution of CHO and fat oxidation to the total energy production were determined [21]. Circulating levels of glucose and lactate were assessed at rest and before the end of six minutes of exercise. Glucose and lactate were

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measured using glucose (Medisafe Mini GR-102; Terumo Co., Tokyo, Japan) and lactate (Lactate Pro2; Arkray Inc., Kyoto, Japan) analyzers. Ratings of perceived

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exertion (RPE) were determined using the Borg 15-point rating scale [22]. ． We established relationships between submaximal intensity and VO2 as well as HR. ． Maximal work rates at estimated maximal HR and VO2 at maximal work rates ． (estimated VO2max) were individually calculated by extrapolating these linear

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relationships. Maximal HR was estimated as 220 minus age [23]. Exercise intensity at ． 50% of the estimated VO2max was individually assumed by interpolating the linear ． ． relationship between work rate and VO2. Exercise intensity at 50%VO2max was ． individually determined from the work rates nearest to 50% of estimated VO2max at the

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three levels of intensity, and then parameters during exercise were subsequently

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analyzed. Data from one participant in the control group who declined to exercise were ． excluded. The day-to-day CV for VO2 during exercise was 5.3 ± 1.5% [17, 20].

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Daily dietary intake and steps Dietary intake before and after the study period was assessed using a brief-type

self-administered diet history questionnaire (BDHQ) [24]. Daily steps were obtained from the health care app on Apple iPhone [25]. According to our studies [26], a valid day was defined as wearing the device for over 2,000 steps, and days when the equipment was not worn were excluded from analysis. We subsequently analyzed data

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about steps from 15 of the participants (Control; n = 7, Intervention; n = 8) who had four or more valid days per week, and data were compared between the first (Pre) and last (Post) two weeks of the intervention period.

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High-cocoa chocolate intervention

Participants in the intervention group consumed 20 g/day (10 g at approximately 10:00 h and 10 g 15:00 h daily; cacao polyphenol, 508 mg) of high-cocoa chocolate

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(Chocolate Kouka Cacao 72%, Meiji Co. Ltd., Tokyo, Japan) for four weeks. Nutrition facts indicate that 5 g of the chocolate included comprised 28 kcal of energy, 0.5 g of protein, 2.1 g of fat, 1.7 g of carbohydrate, 0.6 g of dietary fiber, and 0.3 mg of sodium. Observers provided the same type of chocolate to all participants in the intervention

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group weekly. Daily compliance with chocolate intake was confirmed using the Line social network service app (LINE Co., Tokyo, Japan) via mobile phone. Participants in

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the control group had no concomitant intervention. All participants were advised to

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study period.

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maintain their usual diet and to refrain from any other specific exercise throughout the

Statistics

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The results are presented as means ± standard error of the mean (SEM). Parameters

before the study were compared between the two groups using independent Student t-tests. Changes in parameters were evaluated using a two-way repeated-measures analysis of variance (ANOVA). When the F value was significant, the Bonferroni method was applied for post hoc multiple comparisons. Changes in arterial stiffness between pre- and post-intervention were assessed using independent Student t-tests and

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analyses of covariance (ANCOVA) that included age, sex, height, weight, baseline values, and mean BP as covariates. Relationships were assessed using Pearson correlations. All data were statistically analyzed using SPSS 14.0J (IBM SPSS Japan, Tokyo, Japan) and Excel Statistics 2015 (Social Survey Research Information, Tokyo,

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Japan). Effect size (ES: using Cohen’s d) and statistical power (1 - ) were calculated using G*Power 3. Values were regarded as statistically significant when P < 0.05.

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RESULTS Baseline data

Table 1 shows the physical characteristics of all the participants. None of the parameters significantly differed between the control and intervention groups before the

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study. Estimated HR max in the control and intervention groups was 196 ± 22 and 191 ± ． 15 beats/min, respectively. Estimated VO2max did not significantly differ in either

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absolute (control vs. intervention: 2.35 ± 0.22 vs. 2.43 ± 0.18 L/min) or relative (control

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vs. intervention: 37.8 ± 2.8 vs. 41.6 ± 2.0 mL/kg/min) values per body mass. The work ． rate of exercise at estimated 50%VO2max did not significantly differ between the

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groups (control vs. intervention: 64 ± 6 vs. 72 ± 6 W).

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Effects of high-cocoa chocolate on body composition and hemodynamics Body composition, such as body mass, BMI, body fat, LBM, and waist circumference,

did not significantly change in either group after four weeks (Table 1). Similarly, neither BP nor HR in the supine position significantly differed after the intervention in either group (Table 1). However, two-way repeated-measures ANOVA indicated significant interactions of haPWV and CAVI (both P < 0.05). Mean haPWV (ES, 0.67; 1- 0.70)

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and CAVI (ES, 0.6; 1- 0.61) were significantly reduced (both P < 0.05) in the intervention group (Fig. 2A, C), compared with the control group and even more so after adjusting haPWV or CAVI for age, sex, height, weight, baseline values, and mean BP when analyzed by ANCOVA (Fig. 2B, D). In addition, cfPWV was significantly

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reduced in the intervention (Pre vs. Post: 6.03 ± 0.13 vs. 5.78 ± 0.13 m/s, P < 0.01; ES, 0.81;1- 0.85), but not in the control group (Pre vs. Post: 5.97 ± 0.12 vs. 5.89 ± 0.13 m/s). The changes in arterial stiffness did not significantly correlate with the amount of

= 0.928; cfPWV: r = -0.167, P = 0.537).

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chocolate consumed per body mass (haPWV: r = -0.144, P = 0.297; CAVI: r = -0.025, P

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Effects of high-cocoa chocolate on metabolic parameters at rest and during exercise ． ． ． None of VE, VO2, VCO2, RER, HR, or RPE at rest and during exercise at ． 50%VO2max significantly changed in either group (Table 1, 2). Rates of substrate

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oxidation of CHO and fat and the relative contributions of CHO and fat oxidation to

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total energy production during exercise also did not significantly change in both groups (Table 2). Circulating glucose levels at rest tended to increase in the control groups, and

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significantly increased in the intervention group. Moreover, glucose during exercise significantly increased in both groups after four weeks (Table 2). Lactate during

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exercise also did not change significantly in either group.

Dietary and exercise habits Dietary habits did not significantly change in both groups with the exception of high-cocoa chocolate ingestion. Both groups also stated that their amounts of physical activity had not changed between before and during the intervention period. Daily step

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counts did not significantly change in both groups (control pre vs. post: 7474 ± 672 vs. 7556 ± 797 steps/day; intervention: pre vs. post: 7833 ± 463 vs. 8403 ± 638 steps/day).

DISCUSSION

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The salient findings of the present study were as follows. Four weeks of high-cocoa chocolate consumption reduced haPWV, CAVI, and cfPWV in young healthy individuals, and did not significantly change BP, body composition, RER, and glucose

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at rest and during exercise.

A previous study has found that regular high-cocoa chocolate ingestion simultaneously reduces cfPWV and BP among young healthy individuals [9]. Although PWV is widely identified as the gold-standard among methods of assessing arterial stiffness [6, 27], BP

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is a major confounding factor that has powerful effects on PWV [11, 12]. Thus, we investigated whether regular high-cocoa chocolate consumption directly reduces arterial

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stiffness in young healthy individuals. Our results indicated that the CAVI and PWV

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became significantly reduced after chocolate ingestion. In general, CAVI is an index of arterial stiffness from the aorta to the ankle, after adjustment for BP [28]. In addition,

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BP and HR did not significantly change after intervention. Throughout the study period, neither dietary habits nor physical activity significantly differed throughout the study.

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Therefore, these findings indicated that regularly consuming high-cocoa chocolate reduces arterial stiffness after adjusting BP in young healthy individuals. The physiological mechanisms through which the regular consumption of chocolate with a high cocoa content reduce arterial stiffness remain obscure. However, high-cocoa chocolate can enhance endothelium-dependent vasodilation, primarily through increasing nitric oxide (NO) production or bioavailability [2, 8], which could contribute

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to a reduction in arterial stiffness [27]. Indeed, one study has demonstrated that polyphenols can increase NO synthase activity and NO release in endothelial cells in vitro [29]. Thus, cocoa polyphenol might increase the production or bioavailability of NO, and thus inducing arterial functional changes such as reducing arterial stiffness.

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However, we did not obtain direct evidence in vivo to support this notion and further studies are required.

Acute ingestion of high-cocoa chocolate can increase fat oxidation during exercise [13,

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14]. Although an acute study is important, it was unclear whether regular ingestion of high-cocoa chocolate affects metabolic characteristics at rest or during exercise. After 4 ． weeks of ingestion, none of VO2, HR, lactate, RER, indexes of fat oxidation, body weight, fat, and LBM significantly changed. In addition to the present reduction in

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arterial stiffness, the daily amount of ingested polyphenol in this study was the same or slightly higher than that in previous studies [1-3, 13, 14]. Thus, the results might not

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have been due to an insufficient dose of polyphenol or chocolate. Plasma levels of

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polyphenol or epicatechin, which are components of high-cocoa chocolate, generally reach a maximum at 2 h after intake, and then gradually disappear [30]. Thus, one

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explanation for the conflicting findings between this and previous acute studies might be different circulating amounts of polyphenol or epicatechin at the start of exercise.

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Therefore, the present findings indicate that regular ingestion of high-cocoa chocolate does not affect metabolic characteristics either at rest or during light-to-moderate intensity exercise. Contrary to previous findings [2, 10], we found that resting BP and glucose did not significantly improve in young participants. The magnitude of BP reduction relates to baseline BP levels [10], and young individuals with normal BP are unlikely to have a

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redundant reduction in BP. On the other hand, we also found increased resting glucose levels, especially among those in the intervention group who consumed regular normal diets plus 20 g/day of high-cocoa chocolate. Thus, although body mass and fat did not significantly increase, adding high-cocoa chocolate to the diet might have induced a

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small increase in blood glucose levels, meaning that high-cocoa does not always positively affect metabolic-related health.

The study limitations are as follows. The magnitude of the reduction in arterial

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stiffness was relatively small. However, an increase in PWV of 1 m/s generally corresponded to an approximately 10% increase in risk of cardiovascular events or mortality [6]. Therefore, the effects are clinically noteworthy. Our intervention group was given absolute amounts of chocolate, and the control group did not receive a

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placebo. Alternatively, the changes in arterial stiffness did not significantly correlate with relative amounts per body mass. Moreover, previous studies have not found any

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significant improvements in BP or arterial stiffness after placebo control chocolate

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ingestion [4, 10]. Accordingly, the reduction in arterial stiffness was probably not profoundly affected by minor mass-based differences relative to ingested amounts or

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placebo effects. Finally, we did not measure circulating lipid profiles. However, all participants were young healthy individuals with no chronic diseases, and thus were

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unlikely to have risk factors for atherosclerosis or a need to improve circulating lipid profiles.

CONCLUSIONS Four weeks of regular high-cocoa chocolate ingestion reduced arterial stiffness after adjusting BP in young healthy individuals. However, habitual high-cocoa chocolate

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intake probably does not affect body composition or metabolic characteristics at rest and during light-to-moderate intensity exercise.

ACKNOWLEDGMENTS

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We sincerely thank the study participants for their cooperation. The research did not receive any specific grant from funding agencies in the public, commercial, or

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not-for-profit sectors.

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Fig. 1 Flow diagram of study participants.

CR IP T

volunteers. Free Radic Res 2000;33:635-41.

Fig. 2 Changes in haPWV (A, B) and CAVI (C, D) before and after the intervention.

AC

haPWV, heart-ankle pulse wave velocity; CAVI, cardio-ankle vascular index; Pre, before intervention; Post, after intervention; Control, control group; Intervention, intervention group; ANCOVA, Analysis of covariance. Data are expressed as means ± SE.

19

PT

ED

M

AN US

CR IP T

ACCEPTED MANUSCRIPT

Table 1 Physical characteristics of the participants before and after the intervention. Control group

AC

CE

Variables

Intervention group

t test or ANOVA Interact

Pre

Post

Pre

Post

Group

Time ion

Participants

n = 16 (12/4)

n = 16 (12/4)

(men/women) 20. Age, y

20. ± 0.3

7

-

P= ± 0.3

8

20

-

0.870

-

ACCEPTED MANUSCRIPT

166

164 ± 2.3

-

.7 62. ± 2.7

8

7

22.

22.

21.

21.

± 0.7 3

2

25.

24.

21.

20.

46. ± 2.1

4

8

120

± 7

61

± 3

122

74.

± 1.8

2

± 5

± 4

64

120

CE

AC

Diastolic BP, mmHg

Mean BP, mmHg

Pulse

70

88

± 1

± 2

71

90

± 1

± 2

70

88

1

± 9

± 2

62

± 2

51

± 3

50

120

7.8

± 0.4

7.8

± 0.4

7.8

21

0.453

P=

P=

P=

0.296

0.831

0.523

P=

P=

P=

0.107

0.079

0.336

P=

P=

P=

0.763

0.156

0.917

P=

P=

P=

0.174

0.882

0.632

P=

P=

P=

0.847

0.061

0.608

P=

P=

P=

0.865

0.264

0.487

P=

P=

P=

0.913

0.727

0.647

P=

P=

P=

0.708

0.307

0.473

P=

P=

P=

0.870

0.343

0.653

P=

P=

P=

0.671

0.395

0.489

± 2

± 1

± 1

70

88

± 1

± 1

± 2

50

± 2

mmHg ． Resting VE, L/min

0.851

± 8

pressure, 50

0.233

± 2.0

PT

Systolic BP, mmHg

0

P=

± 2.0

0

74.

± 1.9

ED

64

46.

± 1.9

5

78. ± 1.8

P=

± 1.6

1

45.

± 2.1 8

77.

HR, beats/min

± 1.7 0

AN US

46. Lean body mass, kg

± 2.2 8

P=

± 0.7

4

± 2.0

circumference, cm

± 0.7

3

1

-

± 2.7

4

Body fat, %

Waist

57. ± 2.6

2

± 0.7 kg/m2

0.396

57. ± 2.6

M

Body mass index,

-

.3

62. Body mass, kg

P= ± 1.8

CR IP T

Height, cm

± 0.3

7.4

± 0.4

ACCEPTED MANUSCRIPT

7 ． Resting VCO2,

0.2

1

0.2

0.0

6

2

Resting RER

0.0

Glucose, mg/dL

74

Lactate, mmol/L

1.1

Total caloric intake,

± 3

± 0.1

160

13

79

1.0

4

Carbohydrate intake,

216

20.

217

51.

.9

7

CE

56. 6

9

17. 2

6

± 3

± 0.1

10

0.985

0.663

P=

P=

P=

0.281

0.521

0.606

0.8

P=

P=

P=

0.655

0.227

0.983

P=

P=

P=

0.646

0.008

0.364

P=

P=

P=

0.889

0.008

0.686

14

P=

P=

P=

1

0.588

0.699

0.712

P=

P=

P=

0.376

0.681

0.600

P=

P=

P=

0.590

0.605

0.914

P=

P=

P=

0.835

0.690

0.718

1 0.0

1

82

2

± 3*

1.0

9

8

246

166

20.

6

235

± .5

.7

1

47. ± 3.4

± 4.7 0

57.

57. ± 4.6

2

23. ±

2

48.

± 4.0

± 0.1

±

7

54. 9

0.303

±

1

171

± 5.1

± 5.0

P=

0.0

0

±

50.

± 5.2

Protein intake, g/day

1.1

±

8

PT

Fat intake, g/day

10

ED

.6

75

± 0.1

3

± g/day

± 2

P=

1

0.2

±

± 7

0.0

2

P=

± 1

0.8

1

160

± kcal/day

0.0

1

0.0

0

0.0

5

±

± 1

0.2 ±

1

0.2

1

0.8

± 3

5

0.0

1

0.0 ±

± 1

0.8

0.2

1

0.2

± L/min

0.0 ±

CR IP T

0.0 ±

AN US

0.2

M

． Resting VO2, L/min

± 5.9 1

AC

． ． HR, heart rate; BP, blood pressure; VE, minute expired ventilation; VO2, oxygen ． uptake; VCO2, carbon dioxide output; RER, Respiratory exchange ratio; Pre, before intervention; Post, after intervention; *P < 0.05 vs. each pre. Data are expressed as means ± SE.

22

ACCEPTED MANUSCRIPT

． Table 2 Changes in metabolic characteristics during exercise at 50%VO2max level before and after the intervention. Variables

Control group

Intervention group

ANOVA

Post

Pre

CR IP T

Interac

Pre

Post

Group

Time

tion

1. 6

.6

6

1.

0.

1.

0.

08

09

0.

0.

±

0.

±

± 99 0.

97

0.

0.

± 90

AC %CHO

07 0.

01

1

1.

48

1.

49.

±

0.

0.

0.

0.

1.1

09

91

10

2

0.

0.

0.

0.1

18

±

65

21

3.

57

±

0.289

0.499

0.332

0.

P=

P=

P=

08

0.584

0.112

0.180

0.

P=

P=

P=

07

0.507

0.053

0.416

0.

P=

P=

P=

0

01

0.177

0.090

0.379

47.

1.

P=

P=

P=

4

3

0.927

0.164

0.224

1.0

0.

P=

P=

P=

10

0.356

0.138

0.965

0.

P=

P=

P=

02

0.485

0.333

0.668

3.

P=

P=

P=

5

2

0.177

0.090

0.379

34.

± 3.

P=

P=

P=

1.1 4

1.0

0.0

2

0.9

±

± 1

0.1

±

±

± 02

7

± 1.1 0

±

P=

±

8

0.9

5

0.

0.0

7

.0

Fat oxidation, g/min

P=

±

88

±

P=

±

8

1.0

3

99

0.0

8

01

±

1.

± 6

1.1

.1

CE

CHO oxidation,

48

5

±

PT

． %VO2max, %

0. ±

07

26.

±

08

ED

RER

27. ± 1.8

.4

10 ． VCO2, L/min

1. ±

M

． VO2, L/min

g/min

24

±

AN US

24

． VE, L/min

± 1 0.0

4 0.1

± 03

7

4.

68.

±

± 2

8 65.

± 3.6

contribution, %

.5

6

.1

2

3

%Fat contribution, %

34

± 3.

42

± 4.

31.

23

± 3.6

±

ACCEPTED MANUSCRIPT

.5

6

11

0.

12

± .5

± 3

0.

.0

80

± 2

116

± 3

12.

± 3*

0.177

0.090

0.379

P=

P=

P=

0.540

0.112

0.395

0.

P=

P=

P=

6

2

5

0.856

0.218

0.473

P=

P<

P=

74

0.332

0.001

0.345

P=

P=

P=

0.913

0.709

0.395

± 1

± 3

81

0. ±

1

12. ± 0.4

5

1.

± 5

0. ±

4

1. Lactate, mmol/L

117

2

3

12

69

± 3

5

± 2*

0.

1.6 1

CR IP T

7

Glucose, mg/dL

7

11 ± 2

PRE

2

± 0.1

1.5

±

AN US

HR, beats/min

.9

1

． ． ． VE, minute expired ventilation; VO2, oxygen uptake; VCO2, carbon dioxide output;

M

RER, respiratory exchange ratio; ． ． ． %VO2max, VO2 corresponds to estimated VO2max at each pre; CHO, carbohydrate; CHO and fat oxidation, rates of substrate oxidation of carbohydrate and fat; %CHO

ED

and %fat contribution, relative contribution of carbohydrate and fat oxidation to the

PT

total energy production; HR, heart rate; Pre, before intervention; Post, after

AC

CE

intervention; *P < 0.05 vs. each pre. Data are expressed as means ± SE.

24