The effect of carbohydrate mouth rinse on performance, biochemical and psychophysiological variables during a cycling time trial: a crossover randomized trial

Fourteen male trained cyclists were recruited to participate in the study. Participants were cycling for at least 150 km about 5 h/wk. and were heat acclimated. Exclusion criteria included smoking, previous diagnosis of chronic diseases and musculoskeletal injuries within the last 6 months that could interfere with training routine. The recruitment occurred through the dissemination of the study in digital media and personal contact, in the Department of Physical Education of the Federal University of Rio Grande do Norte and in cycling teams of the city of Natal-RN, Brazil, that meet periodically in several places of the city. This study was approved by the Research Ethics Committee of the Federal University of Rio Grande do Norte under protocol number 31747714.7.0000.5568. All participants were informed about the procedures and signed an informed consent form prior to enrollment in the study. The final number of participants included in the present study was based on the study of Carter et al. [12]. Sample size was calculated with a test power of 90% and significance level of 5%. The protocol of this randomized crossover clinical trial was registered on a publicly accessible database (http://​www.​ensaiosclinicos.​gov.​br/​rg/​RBR-4vpwkg/​).

Each subject attended the laboratory on five different occasions. The first visit aimed to characterize subjects by application of an anamnesis form, collection of anthropometric data and evaluation of performance in an incremental exercise test to exhaustion. The second visit was a familiarization session with the experimental procedures. In the 3th to 5th visits, in random and cross order, the participants completed a 30 km cycle ergometer time trial. All tests were performed in the afternoon and in an acclimatized room, with control of temperature and humidity (average temperature of 22.1 °C and humidity of 72.5%). Participants were instructed to abstain from alcohol, caffeine, tobacco and exercise, and also to stay properly hydrated for 24 h prior each visit. It was mandatory the ingestion of 500 mL of fluid 2 h before the tests to ensure that participants were hydrated, according to the recommendations from the American College of Sports Medicine [24]. All test sessions were conducted at the same time of the day and subjects were in a fed state with a minimum of 2–3 h from the last meal. Participants were also instructed not to change your eating pattern during the study and in the days leading up to the tests, particularly the last meal prior testing. In addition, they were advised to record all foods and beverages consumed throughout the day, including type and amount of food, and time of consumption in a diary. Based on this food diary, nutritional composition was analyzed using the software (DietWin®, 2015).

We evaluated the following anthropometric parameters: body mass, height, and skinfolds. Body mass (kg) and height (m) was determined by a portable digital scale coupled to a stadiometer Welmy® (W 110 H, Santa Bárbara d’Oeste, SP, Brasil), with accuracy of 0.1 kg and 0.01 cm, respectively. These parameters were used to calculate the body mass index (BMI, kg/m²) for classification of the nutritional status according to the cut-off points defined by the World Health Organization [25]. All measurements of skinfolds (triceps, subscapular, chest, biceps, iliac crest, abdominal, medial thigh and calf) were made on the right side of the body by using a compass (Cescorf®) with accuracy of 0.1 mm. Skinfold measurements were used for calculation of body density using the generalized equation proposed by Jackson and Pollock [26] and later converted to fat percentage, according to formula proposed by Siri [27].

Participants performed an incremental exercise test on cycle ergometer (Velotron, Racermate, Inc., Seattle, WA, USA) to determine maximum heart rate (HRmax), peak oxygen uptake (VO₂peak) and peak power output (Wmax). The protocol of test began at 100 W for 1 min followed by fixed increments of 25 W per minute until volitional exhaustion or cadence lower that 80 rpm. Heart rate (beats/minute) was continuously recorded throughout the test using a Polar Monitoring System (RS800cx, Polar Electro®, Oy, Kempele, Finland). Oxygen uptake was continuously recorded using a breath-by-breath gas exchange automatic system (Quark - CPET, Cosmed, Roma, Italy). Wmax was defined as the power output reached during the last full stage completed before volitional exhaustion. HRmax was considered the greater HR achieved during the test [28]. The higher mean oxygen uptake of 30 s during incremental exercise test was defined as the VO₂peak [28].

Participants were under random influence of the following interventions in a fed state: carbohydrate mouth rinse (CMR), placebo mouth rinse (PMR), and drinking “ad libitum” (DAL). The CMR intervention consisted of a solution for carbohydrate mouth rinse comprised 6.4% of unflavored maltodextrin (Atlhetica®), in accordance with recommendations from literature [6]. Subjects received 25 ml of the solution in a cup after every 3.75 km (12.5% of the time trial completed), replicating protocols used in this area of investigation [7, 23, 29]. They were instructed to rinse the fluid around their mouths for about 10 s, and then, spit the whole solution in a container indicated by the investigator. The expelled volume of liquid was measured to ensure that ingestion of solution was not significant. The PMR intervention consisted of a placebo solution prepared from artificial sweetener sucralose in powdered form, concentration of 0.08 g/L. prior to the beginning of protocol, we performed sensorial analysis tests to adjust the taste of the placebo solution, to ensure that the interventions had similar taste. The DAL intervention consisted of water availability as much as the subjects wanted (ad libitum) in cups of 150 mL, and the volume ingested was registered.

Participants performed the 30 km time trial tests using a cycle ergometer coupled to the computer (Velotron, Racermate, Inc., Seattle, WA, USA). Participants were allowed to pedal at a preferred cadence and were encouraged to finish the 30 km time trial in lower time as possible. During the 30 km time trial tests, cadence (rpm), speed (km/h), and power output (W) were recorded by software coupled to cycle ergometer. Further, heart rate (HR, bpm) was monitored using a Polar Monitoring System (Polar RS800cx, Kempele, Finlândia). Every 7.5 Km (25% of the time trial completed), the rating perceived exertion was recorded using scale of RPE proposed by Borg [30]. In the same moment, the affective response was recorded using the Feeling Scale proposed by Hardy and Rejeski [31] for monitor level of pleasure or displeasure during exercise. The time to complete the 30 km time trail tests were recorded by software coupled to cycle ergometer. Participants were not notified about their times to complete time trials in any session throughout the study period as well as they were not informed about performance variables during all time trials tests. In each time trial test, the weight loss (%) was calculated by weight after minus weight before time trail divided by weight before.

Biochemical markers assessments were creatine kinase, insulin and cortisol. Blood samples (10 ml) were collected from an antecubital vein by a trained professional using syringe and disposable needles. Blood samples were collected immediately before, immediately after and 30 min after time trail cessation, in heparin containing tubes and centrifuged at 4000 rpm and 4 °C. Plasma aliquots were stored at − 75 °C to posterior analysis. Biochemical analyses were performed in duplicate using standard commercial kits (Labtest®, Minas Gerais, Brazil). Plasma insulin (uIL/mL) and cortisol (μg/dL) concentration were measured by chemiluminescence (Immulite® 1000 Imunoassay System, Siemens, Germany), with maximum intra-assay coefficient of variation was 4.8 and 7 for repeat samples, respectively. Creatine kinase (U/L) levels were measured by the electroenzymatic method (Labtest®, Minas Gerais, Brazil), with maximum intra-assay coefficient of variation of 6%. For reduce inter and intra-assay variance during analysis, the same laboratory technician perform all analysis. All pipettes were properly calibrated and were pre-wetting tip in the solution to be pipetted. Additionally, before and during the time trial tests every 10 km completed, the glucose (mg/dL) and lactate (mmol/dL) were monitored. For this, blood samples (0.3 μL) were taken from the fingertip and analyzed immediately for blood lactate concentration using reagent strips (Accutrend®, Roche, German).