Participants
Eight young healthy participants (four males and four females; mean age, 24.1 ± 1.8 years; body mass, 64.1 ± 4.2 kg; height, 170.6 ± 5.0 cm; and V̇O2max, 42.4 ± 8.3 ml⋅kg−1⋅min−1) participated in this study. None of the participants had a history of mental or somatic disorder or had engaged in regular endurance training for a minimum of 1 year. This study was performed in compliance with the Declaration of Helsinki. The study protocol was approved by the Ethics Review Board of Ritsumeikan University Biwako-Kusatsu Campus (BKC-IRB-2014-034). Each participant received an explanation of the purpose and potential risks of the study, as well as about the experimental procedure, and provided written informed consent for participation.
Maximal graded exercise test
The participants underwent a maximal exercise test to determine their exercise intensity at least 5 days before the experiment. Oxygen uptake was measured with a breath-by-breath gas and volume analyzer (AE-310S; Minato, Osaka, Japan) during running on a treadmill (Life Fitness, Tokyo, Japan). The participants ran at a constant inclination of 1%, and the speed was increased by 0.9 km/h every 1 min from 6.0 km/h. Heart rate was recorded continuously during the exercise test by using a heart rate monitor (Polar RS800CX; Polar Electro Oy, Kempele, Finland). V̇O2max was considered to be valid when at least two of the following three criteria were met: a plateau in V̇O2
, defined as when V̇O2 at two different grades differs by less than 2.1 ml⋅kg−1⋅min−1 [23], a respiratory exchange ratio of ≥1.10, and attainment of at least 90% of the age-predicted maximal heart rate (220 – age) [bpm] [24].
Experimental protocol
Each participant completed two exercise sessions with two different mouth-rinsing conditions during the exercise: either with a carbohydrate solution (CHO) (Maltodextrin; Body Plus International, Miyagi, Japan) or with water (CON). Participants were blinded to the mouth-rinsing condition. The exercise sessions with different mouth-rinsing conditions (i.e., CHO and CON) were performed at intervals of at least 5 days, with randomized order. Participants were familiarized with the experimental protocols prior to the exercise sessions. For female participants, the experiment was conducted during the follicular phase of the menstrual cycle.
With regards to the participants’ diet, they were supervised to ensure that all of them had the same diets on the days prior to the first and second experiments. On the day before each session, the participants were instructed to have dinner by 22:00 and not consume anything other than water after the meal. On the following morning, the participants visited the laboratory and had breakfast at 8:20. The provided breakfast was identical for all participants and was equivalent to 485 kcal (18.8 g protein, 10.1 g fat, and 75.6 g carbohydrate).
A schematic outline of the present study is shown in Fig. 1. Each exercise session was started 3 h after the breakfast. The participants ingested water (5 mL/kg body weight) before the exercise [25]. Then, each participant ran for 5 min at a workload corresponding to 40%V̇O2max as a warm-up, which was followed by 65-min running at 75%V̇O2max on a treadmill at a constant inclination of 1%. The mean room temperature was 20 °C. Heart rate was recorded continuously during the exercise. Mouth rinsing was performed every 10 min from the start of exercise.
A cognitive function test was administered before exercise (pre-exercise) and after exercise (post-exercise: within 2 min after the exercise session). Immediately before each cognitive function test, subjective perceptions were assessed and a blood sample was taken.
Mouth rinse solution
In CHO, a 6.4% maltodextrin (Maltodextrin; Body Plus International, Miyagi, Japan) (6% carbohydrate) solution was used. Maltodextrin is colorless and unsweetened when dissolved in water. The participants were instructed to rinse their mouths with a mouthful (approximately 25 ml) of the solution for 5 s before spitting into a bowl. The carbohydrate concentration and the volume of the rinsing solution were decided on the basis of previous studies [26, 27].
Executive function test
The modified incongruent Stroop Color and Word Test [28] was administered to assess executive function of the participants. A computer screen that displays different words with different colors was placed 1 m in front of the participants at their eye level in the sitting position. A white fixation cross (+) on a black background appeared for 750 ms followed by word stimulus presentation with a duration adjusted for each participant and a blank black screen for 750 ms. For the incongruent condition, the words “BLUE,” “GREEN,” or “RED” in Japanese were displayed in a different ink color (e.g., BLUE printed in green ink). The congruent version of the Stroop Color and Word Test was used as control condition, in which the words “BLUE,” “GREEN,” or “RED” were displayed in the congruent ink color (e.g., BLUE printed in blue ink). The congruent task is known to measure mainly selective attention and concentration [29]. The participants were asked to identify the color of the displayed word by pressing the appropriate key on the keyboard, with the index, middle, and fourth fingers of their right hand (the dominant hand of all participants). They were then instructed to press the key in accordance with the color of the ink and ignore the semantic meaning as accurately and quickly as possible in the modified incongruent Stroop Color and Word Test. The test consisted of six blocks of 25 trials each, for a total of 150 trials. The trial conditions (congruent and incongruent) were set in a random order.
On the day before the experiment, all participants repetitively practiced reaching the level of >80% of response accuracy [30,31,32] and to reach a stable level of reaction time, to minimize the learning effects. The task difficulty was controlled by adjusting the stimulus duration from 650 to 1000 ms, so that each participant could maintain a > 80% response accuracy.
A software (E-Prime 2.0; Psychology Software Tools, Sharpsburg, PA, USA) was used to present the stimuli and measure the reaction time. The reaction time and response accuracy were averaged for each trial condition. Trials in which the reaction time was recorded to be <120 ms were excluded from the calculation.
Blood analysis
An indwelling needle was inserted into a median cubital vein after breakfast and a 12-mL venous blood sample was taken at each time point. The glucose concentrations were measured by using an automated glucose analyzer (FreeStyle FreedomLite; Nipro, Osaka, Japan). The blood samples were each dispensed into a collection vessel for the analysis of adrenocorticotropic hormone (ACTH), epinephrine, and norepinephrine (NE). All samples were centrifuged at 3000 rpm for 15 min at 4°C. The levels of plasma ACTH, epinephrine, and NE were analyzed by means of reverse-phase isocratic high-performance liquid chromatography (HPLC) at a clinical laboratory (MEDIC, Yasu, Japan).
Subjective measurements
The rating of perceived exertion (RPE) was measured with the Borg 15-point scale, which ranges from 6 (very, very light) to 20 (very, very heavy). Perceptual fatigue, arousal (from extremely high energy to extremely low energy), and pleasure were assessed by using a scale ranging from 1 (not at all) to 20 (maximal). Participants orally responded to these questionnaires before and during exercise.
Statistical analyses
The outcome measures were the reaction time of the Stroop Color and Word Test, the subjective perception, and the blood levels of metabolites and hormones. Data are reported as mean (SD) unless otherwise noted. A two-way repeated-measures ANOVA, with condition (CHO and CON) and time (pre-exercise and post-exercise) as factors, was used to examine the main and interaction effects on the outcome measures. When significant interactions were found, Bonferroni post hoc test was performed to detect the sources of the significant differences. In all analyses, P < 0.05 was used to indicate statistical significance. Partial eta squared (\( {\eta}_p^2 \)) were reported as estimates of effect size. The data were analyzed with SPSS (version 19.0; SPSS, Tokyo, Japan).