Study Design
A randomized, double-blind, placebo-controlled, cross-over design was employed consisting of five separate laboratory visits. During their first visit to the laboratory each participant underwent a familiarization session during which participants were informed of all experimental procedures and familiarized with all performance measures to reduce the possibility of a learning effect. The familiarization trial was identical to experimental trials except that participants consumed no supplement prior to exercise. During the second visit, each participant’s maximal oxygen consumption (VO2max) was determined using a progressive multistage treadmill running protocol.
During subsequent visits, the three main experimental trials consisted of a 5-km running time trial (TT) with cognitive tests before (30 min) and after (+5 min), and were completed in a randomized (www.randomizer.org) counterbalanced sequence separated by 7 days. On experimental days, participants consumed either one (KS1: 22.1 g) or two (KS2: 44.2 g) servings of the ketone supplement (βHB+MCT) or a flavor matched placebo (PLA) drink 60 minutes prior to performing a 5-km running TT on a treadmill. Capillary glucose, lactate, and ketones were measured at baseline, 30 min post supplement ingestion (pre-cognitive test battery), 60 min post supplement ingestion, immediately following the TT (+0 min), and 15 min following the TT (immediately following the cognitive test battery). Other variables that were measured include (a) 5-km running time, (b) RPE (RPE-Overall; RPE-Chest; RPE-Legs), (c) heart rate, (d) affect, (e) session RPE, (f) session affect, (g) 500-m split times during the 5-km TT, (h) and reaction time as well as response accuracy for the Stroop Word-Color Test and Switching Task. RPE, heart rate, and affect were taken every 500-m during the 5-km TT. In addition, during the TT oxygen consumption (VO2), carbon dioxide production (VCO2), minute ventilation (VE) and respiratory exchange ratio (RER), were assessed and derived from indirect calorimetry (Figure 1). Testing sessions were conducted within the Exercise Science Laboratory of Grove City College at the same time each day at a room temperature between 19-21°C and a relative humidity of 35-40%.
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Participants
Thirteen recreational male distance runners participated in this study (Table 1). Participants were recruited directly from local running clubs and by community advertisement. Participants were included if they: 1) completed a 5-km run in under 25 minutes within the last 3 months, 2) were running a minimum of 32 km per week, 3) were between18 and 49 years old, 4) had >2 years of running experience, and 5) were consuming a Standard American Diet [26]. Participants were excluded from the study if they 1) had a history of smoking, 2) had any known metabolic (e.g., diabetes) or cardiovascular disease, 3) presence of orthopedic, musculoskeletal, neurological, psychiatric disorders and/or any medical conditions that prohibit exercise, 4) use of any prescription medications, and 5) following a low-carbohydrate or ketogenic diet. Participants were prohibited from using any ergogenic aids for one month preceding the study and were asked to refrain from taking any performance enhancing supplement(s) during the course of the study. Participants were instructed to refrain from caffeine and alcohol consumption for 48 hours and racing or training for 24 hours, and food and drink for 3 hours before each exercise test. Before enrolling in the investigation, participants were fully informed of any risks and discomforts associated with the experiments prior to giving their written informed consent to participate. The experimental protocol was approved by the Institutional Review Board of Grove City College prior to implementation.
***TABLE 1 Goes here***
Pretrial Preparation
Participants were instructed to maintain their usual training frequency during the study intervention without increasing or decreasing the training load. The participants were instructed to maintain a training log (mode, duration, and intensity of each workout) for one week before the first experimental trial. They were provided with a copy of their pre-trial log and instructed to have the same training routine during the intervention period. In addition, participants were asked to record their training every week during the study (mode, duration, and intensity of each workout). Furthermore, to quantify the subject’s training session intensity, participants were asked to record their session RPE (sRPE) after every training session [26] (pre-trial and within trail), using the OMNI Walk/Run 0-10 Perceived Exertion Scale [27]. Training load for each session was calculated by sRPE x duration of session (minutes) [28]. The sum of each session’s training load provided the quantification of weekly training load. Training load was assessed each week to measure compliance.
Furthermore, participants’ habitual pre-trial and within trial dietary intake was assessed weekly using a 3-day weighed dietary record, consisting of 2 weekdays and 1 weekend day. Participants were provided with a copy of their pre-trial log and instructed to have the same dietary intake during the remainder of the study. During the familiarization session participants were given precise oral and written instructions individually on how to accurately record amounts and types of food and beverages. Participants were provided with a digital portable scale (Ozeri ZK 14-S Pronto, San Diego, CA) and instructed to weigh all food items separately if possible or to estimate the amounts. Diet information was entered into a commercial nutrient analysis software (Nutritionist Pro™, Axxya Systems, Stafford, TX).
Familiarization and Anthropometric Measurements
At the first laboratory visit, all the experimental procedures were explained to the participants. The participants underwent an orientation involving practice of the 5-km TT and familiarization of the cognitive test battery, the various measurement instruments, equipment, affect measures and perceived exertion. Affect was measured using a validated 11-point Feeling Scale [29], with participants informed that their responses should reflect the affective or emotional components of the exercise and not the physical sensation of effort or strain. The OMNI Walk/Run Perceived Exertion Scale [27] was used to measure the physical perceptions of exertion for overall body (RPE-O), legs (RPE-L) and chest (RPE-C). Following the orientation session, anthropometric measures were obtained including height (cm), weight (kg), fat free mass (kg) and fat mass (% and kg). Height (cm) was measured using a physician’s scale (Detecto, Webb City, MO). Participants body mass (kg) and body composition (fat and lean mass) was measured using a Tanita bioelectrical impedance analyzer (BIA) (MC-980Uplus, Tanita Corporation of America, Arlington Heights, Illinois). Finally, the participants performed a 5-km familiarization trial on a motorized treadmill (Trackmaster TMX425C treadmill, Newton, KS).
Maximal Aerobic Capacity
On the second laboratory visit, participants performed an incremental test to exhaustion on a motorized treadmill (Trackmaster). Oxygen consumption (VO2) and carbon dioxide production (VCO2) were measured using an automated metabolic analyzer system (TrueOne 2400, ParvoMedics, Sandy, UT) calibrated prior to each exercise test using standard calibration gases (16% O2 and 4% CO2). Participants wore a Polar heart rate monitor (H10, Polar Electro, Kempele, Finland) during exercise to measure heart rate. After a thorough explanation of the experimental procedures, each participant was instructed to walk on the treadmill for 3 min as a warm-up at a self-selected speed (0% grade). Immediately following the 3-min warm-up, the speed was increased to 5–8 mph for 3 min (0% grade) to achieve the participants’ comfortable running pace. After 3 min of running at 0% grade, the grade was increased 2.5% every 2 min throughout the test protocol while speed was kept constant. The treadmill test was terminated by the subject at the point of volitional exhaustion. At the end of the test, the highest average VO2 value recorded over a 30 second period of exercise was considered the participants VO2max.
Experimental Protocol
On the next three visits, participants were randomly assigned to ingest one of three beverages 60 minutes before the 5-km TT. This timing and dosing strategy is based on our own pilot experiments (unpublished data) showing that capillary R-βHB concentration peaked at 60 min after ingestion of a single bolus of the supplement. The supplement used in this study consisted of βHB-salt+Medium Chain Triglyceride (KETO//OS 2.1 Orange Dream, Pruvit, Melissa, TX, USA). Participants consumed one (KS1: 22.1 g) or two servings (KS2: 44.2 g) of the ketone supplement (βHB+MCT) powder mixed with approximately 237 ml of cold (~6 °C) water. One serving of the supplement contained a calculated 7g βHB racemic salt (50% R-βHB and 50% S-βHB) and reported 7g MCT. A complete list of the ingredients and the relative dose of each ingredient is provided in Table S1. When receiving the PLA, participants consumed an equal amount of water with MiO Orange Tangerine Liquid Enhancer (0 mg caffeine, 0 kcal; Kraft Foods; MiO, Northfield, IL, USA). The ketone supplement, and PLA drink were similar in volume, texture, and appearance. The taste of the drinks was slightly different, and there remains the possibility that the participants were able to identify the drinks. In order to ensure a double-blinded design, each drink was presented to participants in an opaque sports bottle. To avoid the placebo effect in the experimental trials, we did not inform the participants about the names of the drinks and we presented all drinks as having similar ergogenic properties.
Blood Sampling
Fingertip (capillary) blood samples for blood ketones (R-βHB; Precision Xtra, Abbott Diabetes Care Inc., Almeda, CA) and blood glucose (Precision Xtra, Abbott Diabetes Care Inc., Almeda, CA) concentrations were measured at baseline, 30 min post supplement ingestion (30 min; pre-cognitive test battery), 60 min post supplement ingestion (immediately before start of TT), immediately following the TT (+0 min), and 15 min following the TT (immediately following the cognitive test battery). Blood lactate concentration (Lactate Plus, Nova Biomedical) was assessed at baseline, 60 min post supplement ingestion, and immediately post exercise (+0 min). Samples were collected using a lancet following cleaning of the fingertip with an alcohol swab and then dried. The first droplet was wiped away with a cotton swab to remove any alcohol and the subsequent droplets were used for analysis.
5-km Running Time Trial
To determine exercise performance, participants performed a 5-km running TT on a motorized treadmill (TMX425C treadmill; Trackmaster, Newton, KS, USA). Before the start of the run, participants completed a 5-minute self-paced warm-up run. Participants were instructed to finish the run as fast as possible. The gradient was set at 0.0% grade. Participants were provided with feedback on the distance (at regular 500-m intervals) covered during each TT and were not informed of the overall performance time until completion of the study. During the 5-km TT, participants were permitted to adjust their speed how and whenever they saw fit during the TT via control buttons located on the treadmill. The speed indicator and timing devices were concealed from the participant’s view throughout the TT. Therefore, participants regulated their treadmill pace according to their perceived exertion associated with the intensity of the exercise and their subjective feelings of their running capabilities [30]. Heart rate (Polar Electro, Kempele, Finland), RPE (RPE-Overall; RPE-Chest; RPE-Legs) and affect (Feeling Scale) were recorded at 500-m intervals during the 5KTT. Ratings of perceived exertion and affect for the entire exercise session (session RPE and session affect) were obtained 5 minutes following the TT. Metabolic gases were continuously collected during the entire TT using a metabolic cart for assessment of RER, VO2, VCO2, VE, RR, and substrate oxidation.
Cognitive Test Battery
Thirty minutes post supplement ingestion (30 min) and five minutes after each 5-km TT (+5 min), participants performed a battery of cognitive tests to assess executive cognitive function. A familiarization test was performed during the first laboratory visit to reduce the possibility of a learning effect. During this time researchers thoroughly explained the different cognitive tests to the participants, but data were not recorded. Executive function was measured before and after exercise using a computerized automated neuropsychological assessment metric (ANAM®) test (ANAM-4, Vista Life Sciences, USA). The ANAM® is a brief, self-directed, computerized neuropsychological assessment battery that assesses neuropsychological functioning. The ANAM software has been shown to have test-retest reliability [31]. Before each cognitive test battery, time-keeping devices such as watches and cell phones were removed, and during the task, participants did not receive any feedback on performance or time lapsed. Participants were seated in a comfortable chair in a sound-insulated room. Testing was performed under optimal conditions (i.e., appropriate lighting, as quiet as possible, and isolation from unnecessary stimuli). An identical test battery was administered before and after each trial. The battery took approximately 10 min to complete. Participants were instructed to complete the battery as quickly and accurately as possible. For each test, reaction time (in milliseconds) and reaction time for only correct responses (accuracy) were collected.
The test battery consisted of the following validated tests: the Stroop Word-Color Test (congruent and incongruent) and Switching Task (manikin and mathematical processing). The Stroop Test measures cognitive flexibility, processing speed, and executive function [32]. The cognitive mechanism involved in this task is directed to attention, and the participants must manage their attention by inhibiting one response to do something else. For the congruent Stroop Word-Color Test, a series of XXXX’s appeared on the computer screen in one of three colors (“red,’’ ‘‘blue,’’, or “green”). Participants were instructed to press the corresponding key (1 for “red”, 2 for “green” and 3 for “blue” on the keyboard based on color. For example, if the series of XXXX’s appeared in red font then participants were instructed to press 1 on the keyboard. For the incongruent Stroop Word-Color Test, a series of individual words (“RED”, “GREEN”, or “BLUE”) appeared on the computer screen in a color that did not match the name of the color depicted by the word. Participants were instructed to press the response key on the keyboard assigned to the color of the word on the screen. For example, if the word “BLUE” that was written in red ink appeared on the computer screen then participants were instructed to press 1 on the keyboard.
Next, the participants performed the Switching Task. The Switching Task was designed to measure divided attention, mental flexibility, and executive function [33]. The Switching Task requires users to alternate between two tasks: The Manikin and Mathematical Processing. Only one type problem (Manikin or Mathematical Processing) appears on the computer screen. For mathematical processing, participants were presented with a three-digit math equation (e.g., “5+4−2”) and if the sum was greater than “5” they were instructed to click the right mouse, and if the sum was less than “5” then they were instructed to press the left mouse. For the Manikin, participants were presented with an animated character (Manikin) holding a sphere in the left or right hand. If the manikin was holding the sphere in the right hand then participants were instructed to click the right mouse, and if the manikin was holding the sphere in the left hand then participants were instructed to click the left mouse. For each trial, the manikin shifts positions, so that it may be facing towards the viewer, away from the viewer, or to the side. Cognitive test battery was comprised of n = 10 participants for the Stroop Word-Color Test (congruent and incongruent) and n = 11 participants for the Switching Task (manikin and mathematical processing) due to technical issues in n = 3 and n = 2, respectively.
Statistical Analysis
Statistical analyses were performed using SPSS version 24.0 (SPSS Inc., Chicago, IL). Statistical significance was set a priori at p < 0.05. Descriptive statistics were calculated for all variables. Normality and absence outliers were verified by using the Shapiro-Wilk test, normality plots, and residual plots. Performance, physiological, and perceptual data collected during the 5-km TT (5-km running time, mean exercise heart rate, RER, VO2, VCO2, VE, RR, carbohydrate and fat oxidation rates, affect, RPE-Chest, RPE-Legs, RPE-Overall, session RPE and session affect) were analyzed using a one-way repeated measures analysis of variance (ANOVA). A 3 (condition, KS1 vs KS2 vs PLA) × 10 (every 500 meters) repeated measures ANOVA was conducted to assess the effect of time, treatment, and interaction between time and treatment, on heart rate, affect, RPE-Chest, RPE-Legs, RPE-Overall, and time covered at each 500-m interval during the 5-km TT. A 3 (condition, KS1 vs KS2 vs PLA) x 5 (rest, 30 min post ingestion, 60 min post ingestion, immediately post exercise, and 15 min post-TT) repeated measures ANOVA was conducted to assess the effect of time, treatment, and interaction between time and treatment, on capillary glucose and ketones. A 3 (condition, KS1 vs KS2 vs PLA) x 3 (baseline, 60 min post supplement ingestion, and immediately post exercise) repeated measures ANOVA was conducted to assess the effect of time, treatment, and interaction between time and treatment, on capillary lactate. A 3 (condition, KS1 vs KS2 vs PLA) x 2 (time, pre vs post) repeated measures ANOVA was conducted to assess the effect of time, treatment, and interaction between time and treatment, on the Stroop Word-Color Test and Switching Task. A one-way repeated measures ANOVA was used to analyze differences over time for training load and nutrient intake before and during the intervention. The smallest worthwhile change (SWC) was predetermined to be 0.65%. This is midway between the smallest worthwhile change in day-to-day variability in competitive middle distance and distance runners [34] and the estimated coefficient of variation in our laboratory [25, 30, 35]. Post hoc analyses of significant main and interaction effects were conducted where appropriate using the Bonferonni adjustment to determine which conditions were significantly different. The assumption of sphericity was confirmed using Mauchly's test. Greenhouse-Geisser epsilon corrections were used when the sphericity assumption was violated. Partial-eta squared (η2p) was used to report effect size with 0.01 considered small, 0.06 medium, and 0.14 large effects. All data are reported as Mean ± SD.