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CHO Mouth Rinse Ameliorates Neuromuscular Response with Lower Endogenous CHO Stores

ATAIDE-SILVA, THAYS; GHIARONE, THAYSA; BERTUZZI, ROMULO; STATHIS, CHRISTOS GEORGE; LEANDRO, CAROL GÓIS; LIMA-SILVA, ADRIANO EDUARDO

Author Information
Medicine & Science in Sports & Exercise: September 2016 - Volume 48 - Issue 9 - p 1810-1820
doi: 10.1249/MSS.0000000000000973
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Abstract

Carbohydrate (CHO) mouth rinse has demonstrated a positive effect (~3%) on performance during cycling time trial (TT) lasting 30–60 min (6,11). The mechanism governing this improvement is not fully understood, but it is unlikely to be of metabolic origin (6). Instead, it has been proposed that CHO mouth rinse stimulates a group of sensory receptors in the oral cavity (6), which actives some brain areas associated with reward and motor control, including the insula/frontal operculum, orbitofrontal cortex, and striatum regions (8). In support of this assumption, CHO mouth rinse increases motor evoked potentials and improves corticomotor output to the exercised muscles, although this has been demonstrated in a small muscle group (i.e., first dorsal interosseous) (14). Furthermore, a recent study has demonstrated that CHO mouth rinse attenuated cycling-induced (45 min at 70% of maximal power output) neuromuscular fatigue, which may be associated with a better maintenance of corticospinal responsiveness (25). Supporting these findings, another study has also measured the effect of CHO mouth rinse on central motor drive using active muscle EMG during whole-body exercise and found that CHO mouth rinse attenuated the exercise-induced reduction in EMG activity of the vastus lateralis during moderate cycling (80% of respiratory compensation point performed to exhaustion) (2).

A majority of CHO mouth rinse studies have found a positive effect on performance (11). However, some studies do not demonstrate this improved performance, and it is worth noting in these studies that the participants often performed the exercise trial in a fed state (4,39). Consequently, it could be argued that a potential relationship between the preexercise endogenous CHO availability and the exercise performance exists with CHO mouth rinse (8). However, studies investigating this relationship report conflicting results using an overnight fast to manipulate endogenous CHO stores. Lane et al. (26) found that a CHO mouth rinse improved performance during a 60-min cycling TT to a greater extent in the fasted state (10 h overnight fast) compared with the fed state (2 h postprandial). On the other hand, Fares et al. (12) reported similar improvements in endurance capacity with CHO mouth rinse in both fed (3 h postprandial) and fasted (overnight) states. It is postulated that fasting sensitizes CHO receptors in the mouth, consequently stimulating a higher central motor drive and increasing the effect of CHO mouth rinse response on exercise performance (26). Although there is some anecdotal evidence indicating a potential effect of fasting, no experimental study has provided a conclusive direct mechanism to support it.

Liver, but not muscle glycogen, is reduced after an overnight fast (7). Although it remains unknown, it is plausible to suspect that a reduction in the overall level of preexercise endogenous levels of CHO (muscle and liver) may have an added effect on the CHO mouth rinse-induced improvement on exercise performance, when compared with partial endogenous CHO depletion (liver). Although it is yet to be investigated with CHO mouth rinse, evidence from studies of CHO ingestion improving exercise performance leads to presume that preexercise endogenous CHO status would influence the ergogenic benefit of CHO on exercise performance (40). CHO ingestion enabled participants to maintain their optimal pace longer with low initial endogenous CHO availability (i.e., previous exercise at 48 and 24 h before the main trial and a low CHO diet over the 48-h + 6-h fasting) compared with placebo. However, when preexercise endogenous CHO was adequate (i.e., previous exercise at 48 h, not 24 h before the main trial, and a high-CHO diet 48 h prior with only a 6-h fasting), CHO feedings during the exercise did not lead to an additional effect on exercise performance (40). An understanding of the influence of endogenous CHO availability on CHO mouth rinse response is of practical relevance, as CHO mouth rinse has been listed as a candidate, although it has never been tested, to ameliorate the reduced training intensity resulting from low endogenous CHO availability, which is an alternative training approach to maximize mitochondrial biogenesis (1).

Therefore, the main aim of this study was to investigate the effect of CHO mouth rinse on neuromuscular activity (EMG), metabolic responses (plasma glucose and lactate, and fat and CHO oxidation rates), and exercise performance with different starting levels of endogenous CHO availability manipulated by prior fasting and exercise manipulation. Our hypotheses were as follows: 1) the magnitude of improvement on exercise performance with CHO mouth rinse would be inversely related to the extent of preexercise CHO availability, and 2) the mechanism governing the CHO mouth rinse response would be neural rather that metabolic.

METHODS

Participants

Eight healthy, physically active males volunteered to participate in this study. The participants’ age, height, weight, body fat, peak oxygen uptake (V˙O2peak), and peak power output (PPO) were 31.5 ± 7.3 yr, 172.9 ± 5.2 cm, 74.1 ± 11.7 kg, 12.8% ± 4.5%, 46.1 ± 8.2 mL·kg−1·min−1, and 268.8 ± 34.7 W (mean ± SD), respectively. They were all injury free and completed a physical activity readiness questionnaire (Par-Q) elaborated by the American College of Sports Medicine before performing the tests. The human research ethics committee of the Federal University of Pernambuco approved this study, and participants signed an informed consent form before starting the study.

Experimental design

Each participant completed preliminary sessions to ascertain anthropometric assessment (body fat estimation; Jackson and Pollock [23]), a 24-h diet recall, and incremental and a familiarization trial (Fig. 1). Following this, each participant completed six experimental trials in a double-blind, randomized placebo-controlled crossover design. Two experimental trials were performed in a fed state (FED; i.e., 2 h postprandial), two in a fasted state (FAST; i.e., after a 12-h overnight fasting), and two after an exercise-depleting muscle glycogen protocol performed in the evening before the trial, plus a 12-h overnight fast (DEP). The temperature and the relative humidity during the trials were consistent (22.4°C ± 1.7°C, 45.4% ± 7.4% relative humidity).

FIGURE 1
FIGURE 1:
A, Overall experimental design. B, Experimental trial day protocol. IT, incremental test; Fam, familiarization trial; FED, experimental trial in fed state; FAST, experimental trial after a 12-h fasting; DEP, experimental trial after an exercise-depleting muscle glycogen protocol (DEP) and 12-h fasting; PLA, placebo; MVC, 5-s maximal voluntary contraction; b, blood sample; MR, mouth rinse.

Maximal incremental exercise

The incremental test to ascertain V˙O2peak and PPO was performed on a cycle simulator (RacerMate, ComputrainerTM, Seattle [32]). The test consisted of a 3-min warm-up at 50 W, followed by increments of 25 W every 1 min until exhaustion, which was defined as an inability to maintain the pedal cadence between 60 and 70 rpm. V˙O2 and carbon dioxide production (V˙CO2) were measured breath-by-breath throughout the test using a gas analyzer (Cortex Metamax 3B, CortexBiophysik, Leipzig, Germany). The fraction of expired O2 was analyzed with a zirconium sensor and end-tidal CO2 by infrared absorption. Both sensors were calibrated before starting the test with gas containing known concentrations of O2 (12%) and CO2 (5%). The volume of expired air was measured by a bidirectional flow sensor, calibrated with a 3-L syringe.

The V˙O2peak was recorded as the mean V˙O2 values measured during the last 30 s of the test, whereas PPO was recorded as the highest power output (PO) attained during the test. The gas exchange threshold (GET) was established according the following: 1) a disproportionate increase in V˙CO2 versus V˙O2 curve, 2) an increase in the ventilatory equivalent for V˙O2 without an increase in the ventilatory equivalent for V˙CO2, and 3) a first increase in tidal O2 pressure with no drop in end-tidal CO2 pressure (38).

Familiarization session

Participants were familiarized with the experimental procedures of the entire experimental protocol. In chronological order, they performed 1) three sets of a 5-s, one-legged maximum voluntary contraction (MVC), interspaced by 60 s of passive rest; 2) a 30-min constant load (CL) exercise at a workload set at 90% of GET; 3) a one-legged MVC; and 4) a 20-km cycling TT. Exercise models (CL and TT) were designed to last a total of ~60 min, as this has been reported as the optimal duration in which CHO mouth rinse influences exercise performance (6). The CL exercise trial was designed first in the protocol to investigate metabolic and physiological responses in a more controlled environment without influence of power output variations (15), whereas TT was chosen to measure exercise performance because it is a more repeatable performance test and a good reliable test of motivation (27). The configuration of the cycle ergometer was adjusted vertically and horizontally to suit the participant, and the seat and handle bar positions were recorded and then replicated during the subsequent experimental sessions. Cycling shoes were used to secure the feet to the pedals.

Preexperimental trial control

Participants were asked to abstain from all dietary sources of caffeine, alcohol, and strenuous exercise in the 24 h preceding the experimental trials. They were also asked to follow a prescribed set of dietary and/or exercise protocols specific for each trial day. During the first trial, water was provided ad libitum and recorded. The same volume of water was provided in the subsequent trials.

In all preexperimental conditions, the first four meals in the day before the trial were identical. Meal 1 on the day before experimental trial was a breakfast consumed at 8:00 a.m. (485 ± 277 kcal, 63.6% ± 13.1% CHO, 11.7% ± 3.7% protein, and 24.7% ± 9.6% lipid). Meal 2 was a snack consumed at 10:00 a.m. (336 ± 220 kcal, 67.9% ± 22.1% CHO, 7.8% ± 6.8% protein, and 24.3% ± 17.4% lipid). Meal 3 was a lunch consumed at 12:00 p.m. (783 ± 101 kcal, 47.8% ± 10.4% CHO, 28.3% ± 4.1% protein, and 23.9% ± 8.3% lipid). Meal 4 was a snack consumed at 4:00 p.m. (253 ± 155 kcal, 76.4% ± 16.2% CHO, 5.2% ± 3.6% protein, and 18.4% ± 15.5% lipid).

The differences in the exercise and dietary requirements for the various conditions started at the end of the day before the trial (meal 5) and on the morning of the trial day (breakfast). In FED condition, participants had meal 5 at 8:00 p.m. in the evening before and a breakfast at 6:00 a.m. in the trial day. In FAST condition, participants had meal 5 at 8:00 p.m. in the evening before and performed the trial in the next morning after a 12-h overnight fasting. In the DEP condition, the participants attended to the laboratory in the evening before (at 6:00 p.m.) and performed an exercise protocol intended to reduce muscle glycogen content. This exercise consisted of a 90-min CL exercise at 70% of PPO followed by a 5-min rest and then 6 × 1-min intermittent set at 125% of PPO, interspaced by 1 min of passive rest. Participants maintained a pedaling cadence between 60 and 70 rpm during both exercise bouts. This protocol was previously validated to reduce muscle glycogen content by ~50%–70% (16). Meal 5 was offered after the exercise finished (8:00 p.m.), and then participants followed a 12-h overnight fasting until the experimental trial in the next morning.

Meal 5 was an isoenergetic dinner across the three conditions (1082.2 ± 253.3 kcal), but with normal CHO content for FED and FAST conditions (56.4% CHO, 16.9% protein, and 26.7% lipid) and low CHO content for DEP (12.5% CHO, 12.5% protein, and 75.0% lipid). This low-CHO dinner in the DEP condition was offered to prevent glycogen resynthesis after the exercise-depleting muscle glycogen protocol (10).

Experimental trials

Upon arrival at the laboratory, participants rested quietly for 15 min before an intravenous catheter was inserted into the antecubital vein to collect a resting blood sample (1 mL) and enable subsequent serial blood sampling. Before the collection of EMG signal, the skin was prepared, the hair was removed by shaving, the skin lightly abraded to remove the outer layer of epidermal cells, and oil and dirt were removed from the skin with an alcohol swab to reduce skin impedance. Bipolar Ag-AgCl surface electrodes (with interelectrode distance of 20 mm) were subsequently positioned on the vastus lateralis of the dominant leg for EMG record and the reference electrode was placed in a neutral location (bone structure: tibia). The participants were asked to maintain the mark of the electrodes location for the duration of the study, and every time they came to the laboratory, the mark was highlighted as a strategy to keep the electrodes at exactly the same position between the trials. Immediately after EMG preparation, participants performed three 5-s, one-legged MVC of the knee extensors (trunk–thigh at 90° angle and thigh–leg at 0° angle), separated by a 60-s interval. The force produced by the quadriceps muscles was recorded using a load cell connected to a noncompliant strap positioned superior to the ankle (EMG System of Brazil, São José dos Campos, Brazil). The EMG signal was simultaneously recorded with a sample rate of 2000 Hz by a 16-bit A/D converter (EMG System of Brazil). The EMG signal was amplified with octal bio-amplifier with a bandwidth frequency ranging from 20 to 500 Hz (input impedance = 109 ohm, common mode rejection ratio = > 100 dB, gain = 2000), transmitted to the computer and later analyzed with EMG Lab software (EMG System Brazil, São Paulo, Brazil).

After a 5-min resting period, a 30-min CL exercise was performed at 90% of GET. Additional blood samples (1 mL) were collected at 10, 20, and 30 min of the CL exercise. V˙O2 and V˙CO2 were continually recorded, but the mask was taken off for short periods of mouth rinse immediately before and at 5-, 15-, and 25-min exercise. Participants were given a 25-mL bolus of either a tasteless 6.4% maltodextrin (Neonutri-Malto, CHO) or water (PLA), which they rinsed around their mouths for 10 s and then expectorated. Participants were then immediately asked about their RPE (15-point Borg’s scale and RPE) before the mask was reestablished. Raw EMG signal was recorded during 10 s, starting 50 s after the mouth rinse procedure. After the CL, participants moved to the knee-extensor chair, performed one more MVC and withdrew the cannula before performing a 20-km cycling TT. A single MVC rather than three repeats was chosen to provide the fastest possible transition from CL to TT (approximately 5-min separating CL and TT). Reliability data for the use of single knee extensor MVC to assess fatigue have previously been published (17). The mouth rinse procedure (CHO or PLA) was performed before and at the 5-, 10-, 15-, and 18-km mark of TT. RPE and 10-s EMG (starting 50 s postmouth rinse) were also recorded at the same marks.

Blood samples

Blood samples were immediately transferred to vacutainer tubes (Becton Dickinson, BD, Juiz de Fora, MG, Brazil) and centrifuged at 3000 rpm for 15 min at 4°C for plasma separation. Plasma lactate and glucose were analyzed in a UV spectrophotometer (Quimis®, model Q798U2V5, São Paulo, Brazil) using commercial kits (Biotecnica, Varginha, Brazil).

Fat and CHO oxidation rates

The RER and the fat and CHO oxidation rates during the CL (at rest, and 5, 10, 15, 20, 15, and 30 min) and TT (at rest, and 0, 5, 10, 15, and 20 km) were calculated using the mean V˙O2 and V˙CO2 values collected in 1-min intervals. Fat and CHO oxidation rates were calculated using the nonprotein respiratory quotient (13):

in which V˙O2 and V˙CO2 are measured in liters per minute and oxidation rate in grams per minute.

MVC and EMG analyses

Peak force was identified for each contraction, averaged over a 0.5-s period around the peak, and MVC was established as the highest value recorded during the three MVC repetitions. The raw EMG signals were full-wave rectified and filtered with second-order Butterworth band-pass filters with cutoff frequencies set at 10 and 400 Hz to remove external interference noise and movement artifacts. The root mean square (RMS) and the median frequency (MF) for five consecutive contractions during each period of the cycling was calculated, averaged, and normalized by the respective values at the maximal preexercise MVC (19). The MF was calculated from the power spectral density function using a 1024-point fast Fourier transformation (31). These parameters were calculated using the EMG Lab software (EMG System Brazil, São Paulo, Brazil).

Statistical analysis

The data are reported as mean ± SD, unless otherwise noted. A three-way repeated-measures ANOVA was used to verify the effect of preexercise CHO availability (FED, FAST, and DEP), solution (CHO and PLA), and time or distance on dependent variables (glucose, lactate, V˙O2, RER, CHO and fat oxidation rates, HR, RPE, PO, MVC, and RMS). When a significant effect was found, the main effect was identified using Fisher’s significant difference test. A two-way repeated-measures ANOVA followed by a least significant difference test was used to identify the effect of the mouth rinse solution and preexercise CHO availability on exercise performance. As per Batterham and Hopkins (3), the P values obtained from the t-test were used to assess simple effects and make inferences about true (population) values of the effect of CHO mouth rinse on exercise performance. The uncertainty in the effect was expressed as the likelihood that the true value of the effect represents substantial change (harm or benefit). The smallest standardized change was assumed to be 0.20. For all analyses, significance was accepted at P ≤ 0.05. All analyses were performed using SPSS software (version 17.0; SPSS Inc., Chicago, IL), except for uncertainty in the effects, which were calculated using a spreadsheet as described previously (3).

RESULTS

Constant Load Exercise

Plasma glucose and lactate

There was a main effect of preexercise CHO availability on plasma glucose (P = 0.001, Table 1). Plasma glucose was always lower in DEP compared with both FED (P = 0.014) and FAST (P = 0.001) for both CHO and PLA solutions, but there was no difference (P = 0.068) between FAST and FED states. There was also a main effect for time (P = 0.003), with plasma glucose being lower in minute 10 compared with minute 20 (P = 0.016) and 30 min (P = 0.004). There was no main effect of solution (P = 0.210). However, an interaction between solution and time for plasma glucose was found (P = 0.023). Plasma glucose was maintained slightly higher with CHO mouth rinse than with PLA during the exercise. There was no interaction effect between preexercise CHO availability and solution (P = 0.310). However, there was an interaction between preexercise CHO availability and time for plasma glucose (P = 0.011). Plasma glucose increased from rest to the end of exercise during FAST condition but maintained lower and relatively constant during the exercise for the DEP condition. In FED condition, plasma glucose reduced at minute 10, but returned to rest values by minutes 20 and 30.

TABLE 1
TABLE 1:
Metabolic and cardiovascular responses during the CL exercise (90% of the GET) with CHO or placebo mouth rinse after different preexercise endogenous CHO availability conditions.

There were no main effect of preexercise CHO availability (P = 0.060), solution (P = 0.098), and time (P = 0.084) for lactate, but there was an interaction between preexercise CHO availability and time (P = 0.033). Lactate increased from rest to 10 min in all conditions, but this was attenuated in the DEP condition (Table 1).

EMG response

There was a main effect of preexercise CHO availability for RMS (P = 0.005), with values in DEP being lower than both FED and FAST (P = 0.007 and P = 0.012, respectively), but there was no difference between FED and FAST (P = 0.491). There was no effect of solution (P = 0.106) or time (P = 0.064), but there was an interaction between preexercise CHO availability and solution (P = 0.031), with RMS being severely reduced in DEP with PLA, but not in DEP with CHO (Fig. 2A). There were no effect of preexercise CHO availability, solution, time (P = 0.450, P = 0.193, and P = 0.863, respectively), or any significant interaction for MF (Fig. 2B). However, there was a tendency for a significant interaction between the three factors (P = 0.053), with MF being slightly higher in DEP–PLA, but not in DEP–CHO.

FIGURE 2
FIGURE 2:
A, EMG activity (RMS). B, MF of the vastus lateralis muscle (relative to their maximal at preexercise MVC) during CL test (90% of the GET). FEDPLA and FEDCHO, fed with placebo and CHO mouth rinse, respectively; FASTPLA and FASTCHO, fasted with placebo and CHO mouth rinse, respectively; DEPPLA and DEPCHO, endogenous CHO depleted state through preexercise with placebo and CHO mouth rinse, respectively. Values are presented as mean ± SEM. *Significant main effect of preexercise CHO availability (P < 0.05) with DEP lower than FED and FAST. **Significant interaction between preexercise CHO availability and solution (P < 0.05), with EMG activity reduced in DEPPLA, but not in DEPCHO.

Metabolic response, HR, and RPE

There was a main effect of preexercise CHO availability for V˙O2 (P = 0.021), RER (P = 0.001), and CHO and fat oxidation rates (P = 0.001) (Table 1). V˙O2 and fat oxidation rates were higher, and RER and CHO oxidation rates were lower in DEP compared with both FED and FAST. There was no difference between FED and FAST for V˙O2, but fat oxidation was higher and RER and CHO oxidation lower in FAST compared with FED. There was also a main effect of time for all of these variables. V˙O2, CHO oxidation rate, end RER increased, whereas fat oxidation rate decreased, from rest to 5 min and then remained relatively constant throughout the exercise. An interaction between preexercise CHO availability versus time for CHO and fat oxidation rates was also found. The CHO and the fat oxidation rates were maintained relatively constant from minute 5 to the end of exercise in FED, whereas the CHO oxidation rate was reducing and the fat oxidation rate increasing from 5 min to the end of the exercise in FAST and DEP states. There was no significant effect of solution, or significant interaction of solution versus preexercise CHO availability or time.

There was a main effect of preexercise CHO availability for HR (P = 0.005). The mean HR was higher in DEP compared with both FED and FAST (P = 0.017 and P = 0.001, respectively), but there was no difference between FED and FAST (P = 0.927). There was a main effect of time (P = 0.001), with HR increasing quickly in the beginning and thereafter slowly until the end. There were no solution effect (P = 0.883) or significant interactions for HR.

There was a main effect of time for RPE (P = 0.03), with values increasing similarly with time in all three conditions, but there was no preexercise CHO availability (P = 0.677) or solution (P = 0.999) effects or significant interactions.

Force and EMG during MVC

There was no effect of preexercise CHO availability, solution, and time (P = 0.197, P = 0.590, and P = 0.295, respectively) or any significant interaction for MVC before and after CL exercise. There was no effect of preexercise CHO availability and solution (P = 0.317 and P = 0.256, respectively) or any significant interaction for maximal RMS. However, there was a main effect of time for maximal RMS (P = 0.005), with values posttest being lower than pretest. However, there was no effect of preexercise CHO availability, solution, and time (P = 0.248, P = 0.422, and P = 0.443, respectively) or any significant interaction for MF.

The 20-km Time Trial

Performance

There was a main effect for preexercise CHO availability (P = 0.003), with slower performance time for DEP trial (46.34 ± 1.74 min) compared with both FAST and FED trials (42.43 ± 1.43 min and 40.77 ± 1.50 min, P = 0.044 and 0.007, respectively), but there was no significant difference between FAST and FED (P = 0.058) states. There was also a main effect for mouth rinse solution (P = 0.009), with performance time with CHO being faster compared with PLA (42.47 ± 1.39 vs 43.90 ± 1.51 min, respectively). However, there was no interaction between preexercise CHO availability and solution (P = 0.09).

When simple effect was assessed, the performance time was faster with CHO compared with PLA only in the DEP condition (Fig. 3, P = 0.019). The corresponding qualitative inference of CHO mouth rinse was “benefit very likely” for DEP, “possibly benefit” for FAST, and “negligible or trivial” for FED.

FIGURE 3
FIGURE 3:
Performance time during a 20-km cycling TT with CHO or placebo mouth rinse after different preexercise carbohydrate availability conditions. FED, fed state; FAST, fast state; DEP, endogenous CHO depleted state through preexercise; PLA, placebo mouth rinse; CHO, carbohydrate mouth rinse. *Significant faster than PLA in the DEP condition (P < 0.05). Qualitative inference of CHO mouth rinse effect are shown.

There was also a main effect for preexercise CHO availability (P = 0.015), solution (P = 0.027), and distance covered (P = 0.001) on power output during the 20-km TT (Fig. 4A). Power output was higher in FED than that in both FAST and DEP (P = 0.006 and 0.026, respectively), but there was no difference between FAST and DEP (P = 0.211). There was also a main effect of solution (P = 0.027), with power output being higher with CHO compared with PLA. The power output decreased from beginning until km 15, but increased from km 15 to km 20 in all conditions. An interaction between preexercise CHO availability, solution, and distance was also observed (P = 0.001). CHO mouth rinse was progressively attenuating the DEP-induced reduction in power output as distance progressed (Fig. 4A).

FIGURE 4
FIGURE 4:
A, Power output profile. B, EMG activity (RMS) of the vastus lateralis muscle. C, MF (relative to their maximal at preexercise MVC) during the 20-km cycling TT. FEDPLA and FEDCHO, fed with placebo and CHO mouth rinse, respectively; FASTPLA and FASTCHO, fasted with placebo and CHO mouth rinse, respectively; DEPPLA and DEPCHO, endogenous CHO depleted state through preexercise with placebo and CHO mouth rinse, respectively. Values are presented as mean ± SEM, P < 0.05. *Significant main effect of preexercise CHO availability (P < 0.05), with FED higher than FAST and DEP for power output (A) and FAST higher than FED and DEP for EMG activity (B). **Significant main effect of solution (P < 0.05), with CHO higher than PLA. ***Significant main effect of distance (P < 0.05), decreased from 5 to 15 km and then increased. †Significant interaction between preexercise CHO availability, solution, and distance (P < 0.05), with CHO attenuating progressively the reduction of the power output (A) and EMG activity (B) in DEP.

EMG response

There was a main effect of preexercise CHO availability for RMS (P = 0.006), with FAST values being higher than that in both FED and DEP (P = 0.026 and P = 0.015, respectively), but there was no difference between FED and DEP (P = 0.101) (Fig. 4B). There was no distance effect (P = 0.154). A main effect of solution with higher RMS in the CHO than that in the PLA conditions (P = 0.05) was found, which was dependent of the preexercise CHO availability (interaction between solution and preexercise CHO availability, P = 0.010). Similar to the trends in the reported power outputs, CHO mouth rinse had more effect on RMS in DEP (+162%), followed by FAST (+20%) condition (Fig. 4B). There were no effect of preexercise CHO availability, solution, and time for MF (P = 0.955, P = 0.516, and P = 0.956, respectively). However, there was a significant interaction between preexercise CHO availability versus solution versus distance (P = 0.006), with MF reducing progressively with the distance in DEP–PLA but not in DEP–CHO (Fig. 4C).

Metabolic, HR, and RPE response

There was a main effect of preexercise CHO availability for V˙O2 (P = 0.001), RER (P = 0.001), CHO oxidation (P = 0.001), and fat oxidation (P = 0.001). Fat oxidation was higher, whereas V˙O2, RER, and CHO oxidation were lower in DEP compared with both FED and FAST. Fat oxidation was also higher, and V˙O2, RER, and CHO oxidation were lower in FAST than FED. There was a main effect for distance covered (P = 0.001). V˙O2, CHO oxidation rate, and RER increased, whereas fat oxidation rate decreased, from rest to 5 km and then remained relatively constant throughout the exercise. However, there was no effect of solution or interactions for any of these variables (all P > 0.05, Table 2).

TABLE 2
TABLE 2:
Metabolic and cardiovascular responses during the 20-km cycling TT with CHO or placebo mouth rinse after different preexercise endogenous CHO availability conditions.

There was a main effect of preexercise CHO availability and distance for HR (P = 0.028 and P = 0.001, respectively), but there was no effect of solution (P = 0.953). The HR was higher in FED than that in both FAST and DEP (P = 0.048 and 0.037, respectively), but there was no difference between FAST and DEP (P = 0.369). The HR increased from rest to 5 km and then was maintained relatively constant until the end. There was also an interaction between preexercise CHO availability and distance for HR (P = 0.001), with a larger increase from km 15 to km 20 in the DEP condition (Table 2).

There was a main effect of solution and distance for RPE (P = 0.038 and P = 0.001, respectively), but there was no effect of preexercise CHO availability (P = 0.851) or any interaction (P > 0.05). The RPE was slightly lower in PLA than that in CHO during the trial, with values increasing progressively with the distance in all conditions.

DISCUSSION

In the present study, a reduction of preexercise endogenous CHO availability induced by prior exercise and fasting (DEP condition) resulted in a greater physiological stress (i.e., higher V˙O2 and HR and lower plasma glucose) during a set exercise task (i.e., a CL exercise), when compared with postprandial (FED) or partially depleted (FAST) conditions. This elevated physiological stress observed during the CL in the DEP condition was concomitant with a lower EMG amplitude (RMS) in the PLA condition. However, there was a restoration of the EMG activity with CHO mouth rinse, but it had no influence on metabolic responses (plasma glucose and lactate, and fuel oxidation rates). The 20-km TT power output was progressively greater with CHO mouth rinse compared with PLA in DEP state. This was not different in other trials, and it was accompanied with increases in EMG RMS and MF with CHO rinse protocol. When qualitative inference was considered, the CHO mouth rinse effect on the time to complete the 20-km TT was categorized as “benefit very likely” for DEP, “possibly benefit” for FAST, and “negligible or trivial” for FED. Together, these results indicate a centrally mediated response of CHO rinse with improved exercise performance rather than an influence of metabolic mechanisms.

Constant load exercise

An elevated plasma glucose was measured in the first 10 min of CL exercise with CHO mouth rinse (Table 2) and the mechanism by which such an increase occurs early in exercise is unknown. A few studies have evaluated the effect of the CHO mouth rinse on plasma glucose response during TT performances and found no effect of CHO mouth rinse on plasma glucose (9,22,26,34,37,39). However, it is difficult to draw a physiological inference from the TT performance results as a higher power output with CHO mouth rinse reported in those studies (9,26,34) potentially increases muscle glucose uptake. No other study has investigated CHO mouth rinse with CL exercise and, as the exercise power output is constant in this model, the effect of CHO mouth rinse on plasma glucose can be compared. Although expectorated mouth rinse was not weighed to ascertain potential ingestion of CHO, the volunteers were diligent in expectorating on each occasion, and it is unlikely that any considerable amount of glucose could be absorbed directly across the lining of the mouth (21). However, the CHO mouth rinse potentially activates brain areas such as the insula (8), a region that increases sympathetic activity when stimulated (30). Functional studies have demonstrated that such activation of the hepatic and pancreatic sympathetic nerve fibers increases liver glucose output as well as stimulates the release of glucagon and inhibitions of insulin release from the pancreas (24). Nonetheless, the physiological relevance of the ~3% increase in average of the plasma glucose with CHO mouth rinse is unlikely to explain subsequent improvement on exercise performance as no change in CHO oxidation rate was observed.

There was a reduction in the EMG amplitude (RMS) during the DEP condition with PLA mouth rinse. However, the spectral MF was not significantly affected by muscle glycogen depletion, although it seems to be slightly elevated in DEP–PLA condition (Fig. 2B). Although the interpretation of EMG activity during dynamic exercise is difficult, it is the only practical way to measure muscle activation during whole body exercise. A reduction in EMG amplitude has been interpreted as reflective of reduced motor unit recruitment and/or reduced motor unit discharge (28). However, changes in EMG amplitude measuring motor unit recruitment should be interpreted with caution as several factors such as synchronization, prolongation of action potentials, and amount of amplitude cancellation could also influence EMG amplitude (28). On the other hand, an elevation of MF has been interpreted as an increase in the conduction velocity, which has been associated with recruitment of fast-twitch fibers (5,31,35). However, MF during dynamic exercise should also be interpreted with caution because dynamic contractions produce signals that may be nonstationary (28). Apart of this limitation, to the best of our knowledge, only one study investigated the effect of muscle glycogen depletion on EMG activity (both RMS and MF) during a heavy CL cycling (31). These authors found a greater rate of increase in MF, but not RMS, with exercise duration with the heavy CL protocol performed after an exercise regime designed to reduce muscle glycogen of type I muscle fibers. They attributed the increased MF to a recruitment of additional type II motor units to sustain power output as the exercise progressed. Although it is difficult to compare these results with the present study because of differences in exercise intensities (heavy vs moderate, respectively) and muscle glycogen depletion protocols (intentioned to type I vs both type I and II muscle fibers, respectively), it seems reasonable to assume that muscle glycogen depletion alters recruitment strategy regardless exercise intensity. Bearing in mind EMG limitations, our data of reduced RMS might suggest a derecruitment of prefatigued/glycogen-depleted muscle fibers and recruitment of less-fatigued/ partial glycogen-preserved muscle fibers. This may be supported by the slightly increased MF in DEP–PLA condition, suggesting a recruitment of type II muscle fibers. As the force generated by type II motor units is greater than by type I motor units, the fewer motor units may have been recruited to maintain a constant power output (31). This assumption is consistent with a reduction in RMS during the DEP condition compared with the control. In addition, type II muscle fibers are less efficient than type I fibers (31), which is compatible with the higher V˙O2 observed with the CL when starting with reduced muscle glycogen in the present study. However, it should be kept in mind that the level of EMG in other muscles was not monitored, so these assumptions should be carefully considered.

It is interesting to note that the reduction in the EMG amplitude (RMS) during the DEP condition with PLA mouth rinse was ameliorated with CHO mouth rinse. This suggests that the CHO mouth rinse may have activated some brain areas, which might favor a higher motor output (25). Gant et al. (14), using transcranial magnetic stimulation of primary motor cortex, detected an increase in the corticomotor excitability during voluntary muscle activation and maximal voluntary force when nonsweet CHO solutions were present in the mouth. They suggested that afferent signals from oral receptors are integrated with descending motor outputs and might influence muscle force production. It is interesting to note that this effect appeared greater with fatigue, suggesting that the CHO mouth rinse effect may be potentiated when cell energy status is low. Supporting this assumption, Turner et al. (36) showed that oral CHO combined with a motor task increased activation within the primary sensorimotor cortex and the anterior cingulate gyrus. As the anterior cingulate gyrus is a region responsible to drive emotional and behavioral responses to rewarding food stimuli, these authors suggested that exposing CHO in the mouth would be considered high reward value during exercise, given that during intense muscular work fuels become gradually sparse and muscle glycogen is rapidly depleted (36). In a previous study, it was shown that CHO mouth rinse increases RMS during exercise performed at a slightly higher intensity than that used here (80% of respiratory compensation point) (2). The CHO mouth rinse effect on RMS was evident only from minute 30 to 75 min of the exercise, where muscle glycogen is probably reduced and lower than preexercise levels. Our findings expand on these studies by showing that for a given whole body exercise task, CHO mouth rinse ameliorates neuromuscular recruitment when exercise starts with reduced endogenous CHO availability, but not with CHO stores intact or only partially depleted. However, a synchronization influencing RMS or a restored recruitment strategy pattern cannot be discounted. In this regard, evidence suggests that CHO mouth rinse attenuated fatigue-induced decreases on M-wave and motor evoked potential latency (25), which would indicate a preserved neuromuscular transmission and better maintenance of cortical responsiveness, respectively. This is potentially caused by an increased number, and density, of descending cortical neurons ready to fire and the number, and susceptibility, of spinal motor neurons.

Time trial

A reduced pace was observed in the DEP condition for the TT, in which the volunteers were free to regulate their power output aiming to finish the trial as soon as possible. This was not considered to be influenced by the previously performed CL exercise, as the MVC, a general indicator of muscle fatigue (29), was not different before and after the CL exercise in all experimental conditions. The MF measured during MVC was also not altered; however, the RMS was slightly reduced in all three conditions after the CL exercise. It seems improbable for this reduction to be associated with fatigue because if this were the case, RMS would have increased. A reduction in RMS during MVC after CL may be an indicative of an exercise-induced increased neuromuscular efficiency and/or a decrease in the co-activation of antagonist muscles (18). Furthermore, 30 min of cycling at a moderate exercise intensity (90% of GET) is likely to have any homeostatic disturbance and thus minimal effect on subsequent performance (33). Prior exercise and fasting (DEP condition) resulted in an elevated physiological strain (higher V˙O2 and HR) during CL. However, where participants are free to adjust their pace during TT, there was a reduction in these parameters (i.e., lower V˙O2 and HR). This finding indicates that participants chose to reduce their pace during the DEP condition rather than exacerbate physiological stress triggered by a reduced CHO availability.

A lower RMS accompanied the reduced power output in the DEP condition. As opposed to the CL trial, MF was reducing significantly with the distance during the TT in the DEP condition. However, the capacity to apply power output during the trial in the DEP condition was partially recovered with CHO mouth rinse, and this was associated with a concomitant increase in the RMS and MF. A reduced RMS may suggest that muscle glycogen depletion reduces motor unit recruitment and/or discharge rate. Although MF interpretation during nonstationary conditions of the TT is more complicated, a time-dependent reduction in MF with muscle glycogen depletion may indicate a change in muscle recruitment strategy (i.e., derecruitment of type II fibers), which is compatible with a concomitant reduction in power output. A reduction in motor output or change in motor recruitment and, consequently, in external power output might act as a protective mechanism that results from the integration of multiple feedback signals informing a reduced CHO availability in the peripheral tissues (liver and muscle) (1,37). Nevertheless, data in this study suggest that this altered neuromuscular strategy can be ameliorated by the stimulatory effect of the CHO mouth rinse (25). In contrast to CL where participants cannot change the demanding external work, during TT, ameliorated neuromuscular recruitment seems to be converted to a higher power output. Interestingly, Jeffers et al. (25) found that CHO mouth-rinse alleviated neuromuscular fatigue during a 45-min CL exercise, but it did not translate into a subsequent 15-min TT performance improvement. This indicates that a longer TT may be required, compared with the present study (20 km, ~42 min), to accentuate the muscle glycogen depletion and manifest the CHO mouth rinse effect. A large increase in the EMG amplitude and a concomitant increase in the power output with CHO mouth rinse in the DEP condition is affirmation that with greater reductions in CHO stores the more effective the CHO mouth rise protocol (26). These results also support the suggestion that CHO mouth rinse is a potential nutritional intervention strategy to attenuate the reduced training intensity in a low muscle glycogen state (20), which is a training strategy adopted in an attempt to maximize the mitochondrial adaptation to endurance exercise (1). Our results indicate that CHO mouth rinse can provide benefits during a muscle glycogen-depleted state because of a neural modulation with minimal metabolic alteration. Therefore, this intervention would not negatively impact adaptations after low muscle glycogen training, as the metabolic stress on skeletal muscle and the metabolic signaling processes are maintained or greater, inducing similar or enhanced training benefits with reduced muscle glycogen, respectively. Finally, it is likely that CHO mouth rinse will predominantly influence exercise performance with increased exercise duration and as glycogen depletion status reduces. In a real-world scenario, high-performance athletes are often training and competing in a low glycogen status and cannot acutely ingest or use a sustainable amount of CHO during prolonged exercise. This may be the metabolic circumstance when a CHO mouth rinse strategy is beneficial for enhanced exercise performance for greater training adaptation or athletic performance.

CONCLUSIONS

This study demonstrated that reductions of preexercise endogenous CHO availability (with prior exercise and fasting) produced an elevated physiological strain (V˙O2 and HR) and a concomitant reduction in EMG amplitude during a fixed-intensity exercise and reduced power output, EMG amplitude, and EMG frequency during a subsequent 20-km TT. However, CHO mouth rinse restored the EMG amplitude during the DEP condition across both CL and 20-km TT and attenuated the reductions in performance observed. These findings indicate that the CHO mouth rinse effect may be more salient when endogenous CHO status is low.

Thays Ataide-Silva is grateful to the National Council for Scientific and Technological Development (CNPq) for her PhD scholarship. The authors thank all cyclists who took part in this study, and Geivianni Andrade, Marcos Silva-Cavalcante, João Lopes-Silva, Guilherme Ferreira, and Carlos Rafaell Correia-Oliveira for their technical support.

No financial support was received. The authors declare no conflicts of interest. The results of this study do not constitute endorsement by the American College of Sports Medicine.

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Keywords:

CYCLING; EMG; MALTODEXTRIN; MOUTHWASH

© 2016 American College of Sports Medicine