Study design and procedure
This was a double-blinded crossover study divided into three phases, as shown in Fig. 1, and the study was a part of a larger trial. Assessment of health status, measurement of body composition and testing of maximal aerobic capacity were evaluated in phase I. In phase II, the participants performed a high intensity performance cycling session until exhaustion, before nutrition supplementation and a recovery phase of 4 hours, before a new cycling session equal to the first one was performed. Phase III was similar to phase II except for the nutrition supplementation. There were two alternative diets, composed of WP and CHO, with or without supplementation of MPH (CHO-WP-MPH or CHO-WP). The participants could receive either CHO-WP or CHO-WP-MPH in phase II, and the opposite alternative in phase III. The study was conducted at Western Norway University of Applied Sciences from September to November 2017.
Participants
Fourteen healthy male volunteers, with cycling as their main exercise activity, were included in the study. They were recruited through advertising in social media, and from local cycle clubs in Bergen and the surrounding municipalities, Norway.
To avoid hormone differences between individuals, no women were recruited. Eligibility criteria were healthy men between 38 and 55 years of age (changed from 40 to 50 years registered in ClinicalTrials.gov), with a body mass index (BMI) from 19 to 29 kg/m2, who exercised on average between 8 and 12 h per week the last month prior to inclusion, and at least 70% of the exercise had to be cycling. Exclusion criteria were food allergies, self-reported diabetes mellitus, surgery or trauma with significant blood loss or donation of blood within the last 3 months prior to the study. Musculoskeletal problems that could interfere with their ability to perform the cycling sessions were also cause for exclusion. In addition, participants who had human immunodeficiency virus (HIV), hepatitis B surface antigen (HBsAg), or hepatitis C virus antibody (anti-HCV) and/or had been treated with any investigational drugs, steroids, or medications that effected the intestinal function within 1 month prior to the study or use of antibiotics within 3 months prior to the study were excluded.
The study was conducted according to the declaration of Helsinki and the Western Norway Regional Committee for Medical and Health Research Ethics (REK 2017/56) approved the study. Written informed consent was obtained from all participants prior to inclusion.
Intervention and procedures
The participants were instructed to abstain from exercise 24 h prior to the testing in phase I, II and III, and they arrived at the laboratory by car or by public transportation. They were recommended to maintain approximately the same training frequency, volume and intensity between phase II and III, as in the last week before phase II. In addition, they were told not to drink more than five cups of coffee per day during the study period and to refrain from alcohol 48 h prior to each visit.
Phase I
Health status
Health status was assessed based upon a self-reported questionnaire and a further evaluation when necessary as judged by the physician.
Body composition
Height and weight were recorded, including measurement of body composition by use of InBody 720 (InBody Co., Ltd., Cerritos, California, USA). Measurements of body composition included total body weight and height, BMI, fat mass, fat free mass and muscle mass. The BMI was calculated as the body mass divided by the square of height. Measurements were conducted without shoes and socks, and the participants were wearing cycling clothes.
Incremental step exercise test
An incremental step exercise test was conducted on a bicycle ergometer to establish the relationship between workload (Watt/W) and oxygen uptake (V̇O2), and to measure maximal oxygen uptake (V̇O2max) (Jaeger Oxycon Pro GmbH, Würzburg, Germany).
The test started with a warm-up phase at 100 W for 8 min. The workload was then increased by 25 W every 4th min until the blood lactate threshold (LT) was reached. The LT was defined as 1.5 mmol/L above the lowest blood lactate level measured according to methods described by Borch et al. [18]. The cycling was performed with a pedal frequency of 90 revolutions per minute (rpm). Tidal volume (VT), breathing frequency (Bf), V̇O2, carbon dioxide output (V̇CO2) and respiratory exchange ratio (RER) were measured during a period of 60–90 s on each workload. At the end of each workload, heart rate (HR) (Polar Electro OY, Kempele, Finland or Garmin Edge 1000, Garmin Ltd., Schaffhausen, Switzerland) and perceived exertion by use of the Borg RPE scale 6–20 («rating of perceived exertion», RPE) [19] were registered. After each step a measure of capillary blood lactate and glucose were taken from the fingertip and immediately analysed (Biosen C-Line, EKF Diagnostics Holdings plc, Cardiff, United Kingdom).
After reaching a blood lactate level of 1.5 mmol/L above the lowest measure, the test for V̇O2max was performed immediately by increasing the workload with 25 W every 30 s until exhaustion. During this maximal exercise test, VT, Bf, V̇O2, V̇CO2, RER and HR were measured continuously until exhaustion, and at exhaustion the Borg RPE was registered immediately, as well as measurements of blood lactate and glucose.
The participants were cycling either on a Lode Excalibur Sport ergometer (Lode B.V., Groningen, The Netherlands), or on a Velotron bicycle ergometer (RacerMate Inc., Seattle, Washington). Each participant performed every cycling session on the same bike throughout the study. In addition, all individual adjustments for seating position, like the height and angle of the saddle and the handlebar, were identical every time for the same participant.
Gas exchange and ventilatory variables during all cycling sessions were measured using a mixing chamber. The minute ventilation was corrected to the body temperature pressure saturated condition, and V̇O2 and V̇CO2 to the standard temperature pressure dry condition.
Phase II
In phase II, 9–16 days after phase I, the participants performed two high intensity performance cycling sessions with nutrition supplementation and 4 hours of recovery between the sessions.
Phase II involved the following procedures: The participants had a standardized light breakfast meal 1 hour prior to the first high intensity cycling session. Immediately after the cycling session, the participants ingested the nutrition supplementation. After 4 hours of recovery, the cycling session was repeated.
Following both cycling sessions, venous blood samples were taken after 0, 15, 30, 60, 90 and 120 min. At similar time intervals, the participants filled out questionnaires regarding hunger, satiety, abdominal pain, nausea, diarrhoea and desire to eat. Urine was collected during the whole day. Results from these measurements are beyond the scope of this article and will not be presented here.
Two hours into the four-hour recovery period, the participants were served a standardized warm meal. They were allowed to drink a total of 2.5–3 l of water during the day.
High intensity performance cycling sessions
The cycling sessions were initiated with a 20 min moderate intensity at 60% of V̇O2max. The exercise load was then increased directly to 90% of V̇O2max for 5 min. Finally, the participants were cycling on a workload corresponding to 95% of V̇O2max until exhaustion. Linear regression analyses were used to determine the relationship between the workload (W) and V̇O2 measured in phase I, and the W at the given intensities relatively to V̇O2max were further determined based on individual V̇O2max values. The participants were instructed to keep a pedalling frequency of 90 rpm, and exhaustion was achieved when the frequency fell below 80 rpm. The time to exhaustion performed at 95% of V̇O2max was registered. V̇O2, V̇CO2 and RER were measured between 9 and 10 min at 60% of V̇O2max, and between 3 and 4 min at 90% of V̇O2max. HR and Borg RPE were registered every 5 min throughout the cycling sessions, and at exhaustion. Blood lactate concentration and glucose were measured before and immediately after each high intensity cycling session.
The participants were blinded for time to exhaustion when cycling at 95% of V̇O2max.The cycling sessions were supervised by experienced technicians. The participants were informed about rpm during the sessions when needed, but to obtain high test-retest reliability, there was no cheering or encouragement during the cycling sessions.
Nutrition supplementation
Participants reported to the laboratory in the fasted state. They received a standardised breakfast meal comprising a baguette of semi-coarse bread (93 g) with ham (25 g), white cheese (33 g), no butter, coffee (200 mL) and a glass (200 mL) of orange juice, in total 450 kcal and 22 g protein (19.5% (protein energy/total energy) followed by 1 hour rest before the first cycling session. Immediately after the bout, the participants ingested the test or placebo drink, followed by blood sampling (T = 0), and then sampling at intervals for 120 min whilst resting. Then they received a ready-to-use hot meal (Beef Stroganoff with rice, produced by Fjordland, Norway), containing 450 kcal distributed between 57% CHO, 25% protein and 18% fat, whilst resting for another 2 hours before entering the second cycling bout. The participants were allowed to drink a total of 2.5–3 l of water throughout the intervention day.
The nutrition supplementations CHO-WP (placebo) and CHO-WP-MPH (test) were given in the form of a powder dissolved in water. The powders contained 4.2 kcal/gram distributed, in terms of total energy, between 12% from protein, 66% from CHO, and 22% from fat. WP (WPC80/TINE, Norway) was used as the basic source of protein, while the sources of CHO and fat were, respectively, maltodextrin (DE 20) from corn, and vegetable medium chain triglyceride (MCT) powder (BERGAMAST), i.e. MCT coated with maltodextrin at the ratio 70:30, respectively. The powders were slightly acidified with citric acid and flavoured with a strawberry flavouring agent (Firmenich SA, Switzerland) to level out any differences in terms of taste or smell. The serving size of the powders was standardized to 80 kg body weight providing 295 kcal in 70 g of powder giving 3.68 kcal/kg bodyweight, and 20 mg MPH in the test powder equal to a serving size of 1.600 mg in terms of protein (Nx6.25). The placebo powder was made by replacing MPH with equal amounts of WPC80 in terms of protein (Nx6.25) making the powders both isonitrogenous and isoenergetic. By adjusting the amount of powder to their body weight each participant was given equal amounts of MPH-protein or placebo-protein (WPC80) as well as total protein, carbohydrate, fat and energy in terms of body weight. The difference in the amino acid profiles between MPH and WP was considered insignificant. The beverages were made by dissolving powder in cold water at a ratio 1:2 30 min prior to use to form creamy drinks.
The MPH was provided by Firmenich Bjorge Biomarin AS, Ellingsoy/Norway, and was industrially produced by enzymatic hydrolysis of fresh frozen meat from Atlantic cod (Gadus morhua) using the food approved enzyme preparation Protamex® (Novozymes, Copenhagen). The hydrolysate was spray-dried into a powder containing 89% crude protein and < 0.5% fat. The molecular weight (MW) profile of the MPH was analysed by Firmenich-Geneve/Switzerland using size exclusion chromatography (Supradex Peptide 10/300 GL (GE Healthcare, Uppsala-Sweeden)) and UV detection (SEC/UV), and free amino acids by HPLC and Waters Pico-Tag method using UV detection. The analyses showed that about 90% of the peptides had MW less than 2.000 Da (i.e. 18 amino acids or less), about 75% with MW less than 1000 Da (i.e. 10 amino acids or less), and 55% with MW less than 500 Da (i.e. 5 amino acids or less). Twenty-five to 30% of the peptides had MW less than 200 Da representing small dipeptides and free amino acids, the latter accounting for 4.5% of the hydrolysate.
Procedures and blinding
The nutrition supplementations were provided, randomly numbered, from the manufacturer (Firmenich Bjørge Biomarin AS, Aalesund/Norway). An experienced biochemist was responsible for composition and blinding of the diets. In phase II, the participants chose one of two alternative drinks, from identically looking bottles, hereby determining the sequence of the diets. In phase II, five participants chose drinks containing MPH, and nine in phase III. The technicians and the participants were all blinded for the contents throughout the study, and the researchers were blinded during the statistical analyses.
Phase III
The participants returned for crossover testing after a washout period of seven days to repeat the procedures described in phase II. The time of the day was the same for each participant as they met at the same time in the morning in phase II and III in order to avoid circadian variance. The only difference from the protocol was the administration of the alternative beverage.
Outcome measures
Primary outcome in this subanalysis was differences in performance between cycling sessions after diets with MPH compared to diets without MPH, measured by time to exhaustion at 95% of V̇O2max. Secondary outcomes were differences in HR, RER, glucose and blood lactate concentration after diets with MPH compared to diets without MPH.
Statistics
Because less is known about MPH and possible ergogenic effects, compared to indications from previous studies regarding influences of MPH on glucose [20, 21], power estimation in the main trial was calculated based on blood sugar profile. With an estimated change in mean blood sugar profile (area under the curve) of 20%, power of 80%, type 1 error of 0.05 and a standard deviation of 10% the power calculations estimated that 14 participants had to be included in the study.
Descriptive statistics were used to characterize the participants (mean, standard deviation (SD) median and percent). Paired samples t tests were used for comparison between cycling sessions and between the sequences of the nutrition supplementations, CHO-WP versus CHO-WP-MPH (mean, SD and 95% confidence interval (CI)). The outcome variables were differences in cycling time at 95% of V̇O2max, RER measured at 90% of V̇O2max, and HR, glucose and blood lactate measured at the end of the cycling sessions in the morning versus in the afternoon were compared.
We did not ensure equal distribution of CHO-WP-MPH and CHO-WP in phase II and III. However, we found no period or sequence effects on the various outcomes.
The significance level was set at 0.05. The statistical analyses were carried out using IBM SPSS Statistics 24 for windows (SPSS Inc., Chicago, Illinois, USA) and R version 3.4.1 (The R Foundation for Statistical Computing, www.r-project.org).