THE EFFECTS OF ONE-WEEK EXOGENOUS CONSUMPTION ON

TIME TRIAL RUNNING PERFORMANCE

A Thesis

Presented to the

Faculty of

California State Polytechnic University, Pomona

In Partial Fulfillment

Of the Requirements for the Degree

Master of Science

In

Kinesiology

By

Samantha C. Silva

2018 SIGNATURE PAGE

THESIS: THE EFFECTS OF ONE-WEEK EXOGENOUS KETONE CONSUMPTION ON TIME TRIAL RUNNING PERFORMANCE

AUTHOR: Samantha C. Silva

DATE SUBMITTED: Spring 2018

Kinesiology and Health Promotion Department

Dr. Edward Jo Thesis Committee Chair Kinesiology and Health Promotion

Dr. Ken Hansen Kinesiology and Health Promotion

Alexandra Auslander Kinesiology and Health Promotion

ii ABSTRACT

The rationale behind exogenous ketone supplementation is to shift energy substrate reliance, preserve intramuscular glycogen, and improve exercise or sport performance. Prior investigations have demonstrated the ergogenic efficacy of exogenous ketone supplementation, however less is known regarding the effects of a short-term ketone supplementation period on short-distance running time trial (TT) performance in highly-trained subjects. Thus, the purpose of this study was to determine the effects of one-week exogenous ketone salt supplementation on short-distance running TT performance in endurance-trained subjects. In a randomized, double-blind study, endurance-trained male and female participants were allocated to one of the following treatment groups for 8 days following an initial familiarization visit: Ketone supplementation (KET) (n=10) or placebo control (CON) (n=9). Subjects underwent two consecutive (laboratory-based) 800m TT before and after the 8-day treatment period.

Both groups were tested for best and average time-to-completion and blood lactate response during TT performance pre- and post-treatment. There was a significant treatment x time interaction for best TT performance (i.e. fastest time to completion)

(p=0.02). CON demonstrated no change in TT performance from pre- to post-treatment; however, KET improved TT performance as reflected by a 5.8±8.9% decrease in time to completion from pre- to post-treatment (p=0.02, 95%CI= 2.2, 25.2). When controlling for pre-treatment best TT performance, KET had a significantly faster TT than CON

(p=0.03). When examining the average TT performance across the 2 consecutive trials, there was a significant group x time interaction (p=0.04). CON showed no change in average time to completion and KET demonstrated a significant decrease in time to

iii completion from pre- to post-treatment (p=0.04, 95% CI= 0.40-17.2). When controlling for pre-treatment average TT, KET showed a faster TT than CON (p=0.04). Two-way

ANOVA revealed no effect of sex on the pre- to post-treatment change in TT performance (i.e. both best and average performance). Overall, the results support the use of ketone salt supplements as an ergogenic aid for short-distance running performance in trained individuals.

iv

TABLE OF CONTENTS

Signature Page ...... ii

Abstract ...... iii

List of Figures ...... vi

List of Tables ...... vii

Chapter 1: Introduction ...... 1

Statement of the Problem ...... 2

Purpose Statement ...... 2

Significance of the Study ...... 2

Specific Aims ...... 3

Hypothesis...... 3

Limitations ...... 3

Delimitations ...... 4

Operational Definitions ...... 4

Chapter 2: Literature Review ...... 5

Ketone Metabolism ...... 5

Ketone Metabolism under Exercise ...... 7

Exogenous Ketone Supplementation and Human Performance ...... 9

Conclusion and Future Research Implications ...... 10

Chapter 3: Methodology ...... 12

Experimental Design ...... 12

Subjects ...... 13

Dietary Supplementation Protocol ...... 14

iii

Laboratory Testing Procedures ...... 14

Time Trial Testing Procedures...... 14

Physiological Status Monitor Procedures ...... 15

Blood Lactate Measurement ...... 15

Exercise and Dietary Control ...... 16

Analysis of Data ...... 16

Chapter Four: Results ...... 18

Time Trial Performance and Blood Lactate Response ...... 17

Magnitude-Based Qualitative Inference Analysis ...... 20

Chapter Five: Discussion ...... 22

References ...... 29

iv

LIST OF TABLES

Table 1 Between-group comparison of descriptive measures ...... 17

Table 2 Mean values for Pre- to Post-Treatment Change (∆) in Best and Average Time Trial (TT) Time to Completion and Lactate Response during TT .... 21

vi

LIST OF FIGURES

Figure 1 Ketone Metabolism ...... 7

Figure 2 Schematic of Experimental Timeline ...... 13

Figure 3 Pre- to Post-Treatment Change in Best and Average Time Trial Performance ...... 19

Figure 4 Change in Blood Lactate from Rest to Post-TT ...... 20

vii

CHAPTER ONE

Introduction

The optimization of exercise training and human performance has been a hallmark focus of exercise science research (Cox & Clarke, 2014; Cox et al., 2016; Egan

& D'Agostino, 2016b; Evans, Cogan, & Egan, 2017; Zajac et al., 2014). Within this line of investigative literature, various ergogenic strategies have been explored, especially those pertaining to nutritional modifications. Recently, ketogenic diets have received increased attention with a concomitant level of scrutiny regarding its efficacy in enhancing endurance performance. The theory underlying the use of high-fat and low programs by athletes involves the purported improvement in the use of lipids and as an energy substrate in efforts to preserve muscle glycogen (i.e. muscle “fuel”) during exercise or physical competition. However, prior research, such as Burke et al. (2017), Fleming et al. (2003), and Zajac et al. (2014), demonstrates its futility as results reported negative outcomes for performance or training adaptations. Ketogenic diets have shown to improve fat oxidation capacities and provide alternative energy substrates in the form of ketone bodies/ but ultimately rely on the depletion of muscle and hepatic glycogen which would counteract exercise performance (Evans et al., 2017; Laffel, 1999; Robinson & Williamson, 1980).

Exogenous ketones, namely beta-hydroxybutyrate esters or salts, have recently been implicated as an alternate and perhaps more effective means of providing energy substrate support to aid in muscle glycogen preservation during exercise and thereby performance. A body of research, albeit limited, have demonstrated the efficacy by which ketone supplementation aids athletic performance through an integrative

1 mechanism of energy substrate provision and muscle glycogen preservation while also showing some therapeutic benefits (Cox & Clarke, 2014; Cox et al., 2016; Evans et al.,

2017). What remains uncertain are the effects and applications of long-term or prolonged ketone salt supplementation on short-distance running performance, especially in highly trained individuals.

Statement of the Problem

The rationale behind the use of exogenous ketones is to aid human performance through “alternative” (i.e. not lipid, carbohydrate, or amino acids) energy substrate support. Prior research has shown improvements in cycling performance and even increased muscle glycogen resynthesis during recovery with consumption of a ketone ester drink (Cox et al., 2016; Holdsworth et al., 2017). There are several questions that have yet to be elucidated regarding ketone supplementation and human performance can a relatively long-term ketone supplementation period 1) enhance short-distance running time-trial performance, 2) decrease reliance of carbohydrate energy substrates, or 3) improve fatigue resistance during repeated exercise bouts?

Purpose Statement

The purpose of this study was to determine the effects of one-week exogenous ketone salt supplementation on short-distance running time-trial performance in male and female endurance-trained subjects.

Significance of the Study

The results of the proposed study may further add to the body of scientific and practical information on the use of dietary ergogenic aids and specifically the efficacy by which exogenous ketones may be utilized by athletes and fitness enthusiasts alike. The

2 data gathered from this study would be valuable for competitive athletes and all exercising populations who are pursuing various strategies for enhanced exercise performance through nutritional manipulation. This study is one of the first, to our knowledge, to examine the effects of ketone salts on high intensity, short-distance performance in endurance-trained subjects. Lastly, the results of the study may help advance the understanding of ketone body metabolism especially during exercise.

Specific Aims

Aim 1: To determine the efficacy by which a one-week ketone salt supplementation period improves performance on a laboratory-based 800m running time-trial test.

Aim 2: To determine the effects of a one-week ketone salt supplementation period on consecutive 800m running time trial performance and fatigue resistance.

Aim 3: To examine the effects of a one-week ketone supplementation period on activity of anaerobic glycolysis during exercise via blood lactate measurements.

Hypothesis

Following one week of ketone salt supplementation, running performance and fatigue resistance will improve in comparison to a placebo control treatment.

Limitations

The potential limitations of the proposed study included the following: 1) the protocol employs an exercise which may or may not require unaccustomed movements for participants, however an initial familiarization visit will be implemented to address this limitation; 2) a potential learning effect may exist between pre- and post-treatment performance testing visits, however an initial familiarization visit will be implemented to

3 address this limitation; and 3) normal dietary intake will be self-recorded by participants for the duration of the study.

Delimitations

This study was delimited to healthy, endurance-trained male and female subjects between the ages of 18-29 years. Participation will be denied if the subject reports a medical history that would contraindicate the protocol or confound the results.

Operational Definitions

Endurance Trained — Individuals that have performed endurance exercise at least 3 days per week 1 hour in duration for 6 months prior to the start of the study.

Ketogenesis — Occurs largely during starvation, fasting, prolonged exercise, and nutritional manipulation and is stimulated primarily by decreased hepatic glycogen.

Exogenous — Originating or produced outside of the body.

Ergogenic Aid — Any substance, process, or procedure that may, or is perceived to, enhance performance through improved strength, speed, response time, or the endurance of the athlete.

4 CHAPTER TWO

Literature Review

Introduction to Ketone Metabolism

Historically, dietary carbohydrate (CHO) and fat intake have received continuous

attention and scientific scrutiny as it relates to their role as an energy substrate or “fuel”

for active skeletal muscles during exercise. A relatively large body of scientific work has

been specifically dedicated to uncovering the contributions of these fuel sources during

competitive and recreational exercise, alike. Ultimately, specific manipulations in the

intake strategy of these macronutrients have shown to directly influence exercise

performance and thus, have become a central component of an athlete’s nutritional

programming. Besides CHO and fat, an alternative energy substrate known as ketone

bodies or ketones has recently received growing attention in scientific and sport

communities.

Ketones are lipid-derived organic compounds that serve as an energy substrate for extra hepatic tissues especially during low exogenous and endogenous CHO availability

(Laffel, 1999). In a post-absorptive CHO depleted state, endogenous ketone production, or ketogenesis, will increase concomitantly with fat oxidation to supply energy substrates for various cells in the body, especially those that cannot derive energy from fat like the brain

(Evans et al., 2017; Laffel, 1999). Plasma ketone concentrations reflect the ratio between ketogenesis and ketolysis with normal levels relatively low (0.1-0.5 mmol/L) (Féry &

Balasse, 1986; Laffel, 1999; Robinson & Williamson, 1980). Hyperketonemia is marked by plasma concentrations exceeding 0.5 mmol/L while prolonged fasting or starvation may increase plasma ketone levels up to 10 mmol/L for approximately 72 hours (Balasse

5 & Féry, 1989; Biden & Taylor, 1983; Laffel, 1999; Owen et al., 1973; Robinson &

Williamson, 1980). The increase of ketones under a CHO depleted state (such as during fasting) is in a sense an innate survival mechanism to supply the central nervous system

(CNS) and peripheral tissues like muscle with energy substrate for the eventual formation of adenosine triphosphate (ATP), the cell’s energy currency. The primary ketone bodies are acetoacetate (AcAc) and beta-hydroxybutyrate (ß-HB); however, is also technically a ketone body but is generally excreted or expired. Ketones are synthesized through a series of enzymatic, sequential reactions, beginning with the condensation of acetyl-coenzyme A (Ac-CoA) and acetylacetate-coenzyme A (AcAc-CoA) eventually resulting in the release of AcAc. (Figure 1). Once released into circulation, AcAc is transported into extra-hepatic tissues through monocarboxylate transporters (MCT) and is oxidized in the mitochondrial matrix of the cell, initiating ketolysis and subsequent oxidation which eventually leads to the release of chemical energy to form ATP (Evans et al., 2017; Laffel, 1999; Newman & Verdin, 2014). A rise in circulating ketones has shown to result in a subsequent rise in ketone oxidation rates by skeletal muscle (Robinson &

Williamson, 1980). Thus, skeletal muscle may effectively utilize ketones as an

“alternative” energy provision when CHO stores (i.e. glycogen) are limited.

6

Figure 1. Ketone Metabolism – adapted from (Evans et al., 2017) Free fatty acids (FFAs), fatty acyl CoA (FA-CoA), acetoacetyl CoA (AcAc-CoA), Ac- CoA acetyltransferase (ACAT), hydroxymethylglutaryl-CoA (HMG-CoA), hydroxymethylglutaryl CoA synthase (HMGCS), monocarboxylate transporter (MCT) , HMG-CoA lyase (HMGCL), β-hydroxybutyrate (βHB), 3-hydroxybutyrate dehydrogenase (BDH), Ac-CoA acetyltransferase (ACAT), succinyl-CoA:3-oxoacid CoA transferase (OXCT). Protein content and enzyme activity that are higher in exercise- trained skeletal muscle are indicated by the orange circle.

Ketone Metabolism during Exercise

Substrate utilization during exercise is highly dependent on exercise intensity, nutritional intake, and training status (Cox et al., 2016; Egan & Zierath, 2013; Evans et al., 2017; Laffel, 1999). During high intensity exercise (e.g. 75% VO2 max), skeletal muscle predominately utilizes CHO substrates like and especially intramyocellular glycogen. Under moderate and lower intensities, lipids/fats (i.e. fatty acids) may be increasingly oxidized along with CHO sources in skeletal muscle for the production of ATP. Only under particular metabolic stresses to skeletal muscle, lactate, protein/amino acids, or ketones are utilized as energy substrates. As indicated above, ketogenesis and subsequent muscle ketone oxidation primarily occurs during a CHO

7 depleted state, which may be provoked by nutritional manipulation or fasting (Egan &

D'Agostino, 2016b). Ketogenic diets consisting of high fat (>80% of total caloric intake), low CHO (~20%), and moderate-low protein (~15%) increase the reliance on fat oxidation for energy support due to low CHO provision and subsequent endogenous

CHO depletion (Evans et al., 2017; Laffel, 1999; Robinson & Williamson, 1980). With an increase in fat oxidation, there is a commensurate rate of increase in ketogenesis raising plasma ketone concentrations upwards to 7 mmol/L (Evans et al., 2017;

Pinckaers, Churchward-Venne, Bailey, & van Loon, 2017).

In the context of athletic performance, ketogenic diet programs have been adopted to elicit a metabolic adaptation in which the body, in particular skeletal muscle, becomes more efficient in utilizing fat and ketones as an energy substrate (Cox et al., 2016; Evans et al., 2017). However, this method has produced mixed results regarding performance enhancement based on the body of scientific literature. Nutritional , such as through ketogenic dieting, is permissible during a CHO depleted and therefore low muscle glycogen state (Cox et al., 2016; Evans et al., 2017). It has been suggested that regardless of an enhanced capacity for fat and ketone utilization during ketogenic dieting, depletion of muscle glycogen would not be conducive to athletic ability and performance

(Cox & Clarke, 2014; Urbain et al., 2017). For instance, studies by Johnson and Walton

(1972), Fleming et al. (2003), Zarjac et al. (2014), and Urbain et al. (2017) showed inhibited performance capacities following a ketogenic diet in untrained and trained subjects (Fleming et al., 2003; Urbain et al., 2017; Zajac et al., 2014). Moreover, healthy adults demonstrated a negative impact on peak power, endurance capacity, and time to exhaustion following 6 weeks of ketogenic dieting (Urbain et al., 2017). These effects

8 have been attributed to the glycogen depleting effects of the diet and the extensive time to keto-adaptation (Volek, Noakes, & Phinney, 2015). Even in a keto-adapted state, the exercise economy of elite race walkers was impaired during a 3-week ketogenic diet.(Burke et al., 2017).

Well-trained individuals, however, have demonstrated a greater capacity to utilize ketones following a ketogenic diet due to increased enzymatic activity and metabolic clearing rates (Adams & Koeslag, 1988; Evans et al., 2017; Vogt et al., 2003).

Regardless, prior research on nutritional ketosis in highly trained athletes remain inconclusive. Again, it is speculated that CHO depletion would render a large limitation to one’s performance capacities despite the improved ability to oxidize fat and ketones.

However, given the value of ketones as an energy substrate for active muscles during exercise, a strategy to raise plasma ketone availability while maintaining sufficient CHO stores would conceivably support exercise bioenergetics and thereby performance. Thus, the use of exogenous ketones via dietary supplementation compared to other nutritional approaches may serve as a more efficacious means by providing alternative energy provision while preserving muscle glycogen levels.

Exogenous Ketone Supplementation and Human Performance

The utilization of ketone bodies without depletion of glycogen stores is the foundation of exogenous ketone supplementation. Ketosis has been theoretically shown to increase energetic efficiency as well as spare fuel and may provide a means to extend human performance. (Cox & Clarke, 2014). Although the literature on exogenous ketone use remains sparse, a comprehensive study by Cox et al. (2016) has demonstrated the efficacy of ketone ester supplementation in raising plasma ketone (ß-HB) levels in a non-

9 fasted or ketogenic state demonstrating adequate absorption of the exogenous ketones.

Cox et al. (2016) reported ingestion of 573 mg/kg bodyweight of ketone esters increased circulating ketone levels to a similar level following an overnight fast. In addition to these effects on plasma ketone levels, the exogenous ketone treatment altered energy substrate metabolism at various exercise intensities. Data indicated a decline in plasma ß-

HB concentrations across steady state exercise indicating the possibility of alterations in substrate utilization when excess ketones are in circulation. An analysis of respiratory gases supported this contention of increased utilization of ß-HB during exercise. In the same study, exogenous ketones enhanced endurance exercise performance in highly trained cyclists (Cox et al, 2016). Evans et al (2017) noted that the reported contributions may be due to the highly trained status of subjects used by Cox et al (2016). Highly trained populations have shown greater capacity to oxidize ketone bodies (Evans et al.,

2017). The current research pool, albeit limited, has demonstrated the possibility of a greater capacity to utilize ketones without limiting nutritional intake to a ketogenic diet.

What has yet to be determined are the effects of long term ketone salt ingestion on human performance. Most of the current literature has focused on ketone esters as a more suitable exogenous approach than ketone salts due to their purported stability in digestion and absorption (Evans et al., 2017). However, ketone esters remain relatively expensive to produce and purchase, so ketone salts may be a more pragmatic option for general consumers.

Conclusion and Future Research Implications

In conclusion, endogenously produced ketones may serve as an energy substrate during fasting or in a CHO depleted state, but have shown limited potential in furthering

10 human performance. However, the use of exogenous ketone supplements may be an effective means to enter ketosis without large dietary modifications and to replete muscle glycogen stores during exercise. A limited body of research point towards the efficacy of exogenous ketones as a dietary ergogenic aid however, what remains unclear is the efficacy by which ketone salt supplementation enhances higher intensity, shorter duration running performance and fatigue resistance during repeated high intensity exercise bouts in highly trained athletes.

11 CHAPTER THREE

Methodology

Experimental Design

A between-group, double-blind, placebo-controlled experimental design was executed for the proposed study. Participants visited the Human Performance Research

Laboratory (43-202b) at California State Polytechnic University, Pomona for three separate visits. The protocol was administered by the same investigator(s) for each subject for all visits. The first visit served as a familiarization visit followed by two experimental testing trials separated by exactly one week. During the familiarization visit, participants signed an Informed Consent Form and completed a health and exercise questionnaire to determine eligibility. Then, participants underwent laboratory testing for anthropometric measures (i.e., weight and height), body composition, and resting blood pressure. Afterwards subjects were familiarized with the repeated 800m time trial protocol (described below). Subjects were then randomly assigned to one of the following experimental groups in a counterbalanced manner: KET (n=10) or CON (n=9).

The KET group consumed the experimental ketone salt supplement while CON consumed an isocaloric placebo for 8 consecutive days.

For the pre- and post-treatment testing visits, subjects reported to the laboratory after a 3-hour fast. In addition, subjects will be instructed to refrain from any exercise 24 hours prior to each experimental trial and to wear similar exercise apparel to each visit.

The subject initially rested for 10 minutes. During this time the subject was fitted with a wireless physiological status monitor (PSM) and subsequently prepared for time trial procedures. Afterwards, subjects underwent two consecutive 800m running time trials on

12 a self-propelled, motor-less treadmill (TrueForm Runner; Chester, CT) with each trial separated by 5 minutes of active recovery (low intensity treadmill walking). During this test, subjects were blinded to all performance and physiological metrics. Blood lactate measurements were taken during rest prior to the time trial, immediately after the second bout, and 5 minutes thereafter.

Figure 2: Schematic of Experimental Timeline.

Subjects

Nineteen (n=19) healthy college aged, endurance-trained male (n=11) and female

(n=8) were recruited for this study (age, 23.5 ± 2.0y; 168.6 ± 11.8cm; 66.9 ± 13.0 kg,

23.4 ± 2.7 kg/m2) from a convenience sample of collegiate endurance athletes. Each volunteer was required to complete a pre-participation screening, health history questionnaire and provide written informed consent prior to participation. Volunteers met the following inclusion criteria to participate: 1) aged 18 to 29-years, and 2) endurance- trained runners (i.e., high intensity endurance running is performed at least 3 days per week at least 1 hour in duration for 6 months prior to the start of the study). Subjects were excluded from participation if they report or exhibit: 1) a history of medical or surgical events in which the study protocols would be contraindicated or confound the

13 interpretation of results. These include, but are not restricted to, cardiovascular, metabolic, pulmonary, renal, or kidney diseases, hypertension, or musculoskeletal impediments; 2) use of any medication including those with cardiovascular, pulmonary, thyroid, hyperlipidemic, hypoglycemic, hypertensive, endocrinologic, psychotropic, neuromuscular, neurological, or androgenic implications; 3) pregnancy; and 4) daily use of ergogenic aids or dietary performance supplements within 6 weeks prior to the study.

Subjects were asked to maintain normal physical activity/exercise levels during the study timespan.

Dietary Supplementation Protocol

KET and CON received 8 servings of a ketone salt supplement and placebo, respectively. Both groups consumed 1 serving of their respective supplement each day for 8 consecutive days. A serving was provided by the investigator following completion of Visit 2 and 30 minutes prior to testing during the last visit (Visit 3). All supplements were administered in liquid form (powder mix) and matched for caloric value, taste, and appearance. An outside member naive to the nature of this study prepackaged each serving of the assigned supplement in packages labeled “O” or “W” representing either

KET or CON conditions. Information indicating the content of packets “O” or “W” was sealed in an envelope and opened once data collection was completed. The ketone salt supplement contained a proprietary blend of calcium, 11.7g of sodium beta hydroxybutyrate (ketone salt), 100mg of caffeine, 20g of carbohydrate, 130g of sodium,

95mg of potassium. The placebo was a similar mixture minus the ketone salts.

Physiological and Performance Assessments

14 Running Time Trial Protocol. To measure performance, subjects performed two consecutive 800m time trial (TT) bouts on a self-propelled, motor-less treadmill

(TrueForm Runner; Chester, CT) during pre- and post-treatment visits (Visits 2 and 3, respectively). Prior to time trial testing, subjects were given 5 minutes to stretch and 5 minutes to warm up (< 5 mph) on a treadmill (Startrac, Irvine, CA). Then the subjects were positioned on the motor-less treadmill and prompted to begin running towards their desired pace. Once subjects achieve their desired running form and pace they verbally signaled the researcher to begin the TT. Upon signaling, the researcher simultaneously initiated the timer and computerized distance recorder interfaced to the treadmill. The subjects were then given verbal encouragement throughout the 800m TT as well as a verbal cue at 400m, 600m, and 700m. Following each TT bout, subjects performed 5 minutes of light treadmill walking (<5 mph) for active recovery. Prior investigations have demonstrated the reliability of utilizing a non-motorized treadmill to measure performance (Stevens et al., 2015; Waldman, Heatherly, Waddell, Krings, & O'Neal,

2017). These prior investigations have demonstrated the need for a familiarization visit to reduce the learning effect on participants. Therefore, the first visit served as familiarization and the subsequent visits served as pre- and post-supplementation testing sessions.

Physiological Status Monitor Procedures. Prior to the TT protocol, subjects were fitted with a wireless physiological status monitor (PSM) (Zephyr Technology,

Bioharness 3, Annapolis, MD). The PSM collected heart rate, ventilation rate, and electrocardiograph (ECG) data for the duration of the time trials. Wireless data acquisition was performed using a mobile application (IoTool version 1.0.17415;

15 Solvenia). Raw data for each measure was uploaded to an online cloud server after each test.

Blood Lactate Measurement. Blood lactate was measured using a portable blood lactate monitor (Nova Biomedical, Lactate Plus, Waltham, MA) and blood collection via a finger prick method. The fingertip was cleaned with isopropanol alcohol and allowed to air dry before a sterile, single-use lancet was used to initiate blood collection. The first droplet was wiped away with a paper towel and the subsequent droplets was used for analysis. Blood samples was collected and measured at rest and immediately after the second TT.

Exercise and Dietary Control

Subjects were instructed to maintain normal dietary intake and exercise activity for the duration of the study and asked to avoid consuming any other ergogenic aids or supplements. Dietary intake will be monitored through the MyFitnessPal (Under Armor

Inc., Baltimore, MD, USA) mobile application which was used to record daily food and nutrient intake and estimate calorie consumption. There were no between-group or within-group changes in total daily caloric, carbohydrate, fat, or protein intake.

Analysis of Data

A 2 (time) x 2 (group) x 2 (sex) multifactorial Analysis of Variance (ANOVA) was used to identify main effects and/or interactions. In the event of a significant interaction, pairwise comparisons were made using the Bonferroni post-hoc test.

Additionally, a between-group comparison of post-treatment dependent measures was performed using an Analysis of Covariance (ANCOVA) with pre-treatment measures as a covariate. All tests of interaction and main effects were performed using Statistical

16 Package for Social Science (SPSS, version 14.0 Chicago, Illinois) with significance set at p<0.05. The effects of treatment were calculated as the percent change in TT performance and TT lactate response from pre- to post-treatment between KET and

CON. Magnitude-based inference analysis, as described previously (Batterham &

Hopkins, 2006; Hopkins, Marshall, Batterham, & Hanin, 2009), was used to identify clinically meaningful differences in the delta score of each performance measure between treatment groups. The precision of the magnitude inference was set at 90% confidence limits, using a p-value derived from an independent t-test comparing mean differences in percent delta change of performance measures between groups. Threshold values were standard deviations of control group values multiplied by 0.2. Inferences of true differences between CON and KET were determined as beneficial (positive), trivial, or harmful (negative) (Batterham & Hopkins, 2006). Inferences were based on the confidence limit relative to the smallest clinically meaningful effect to be positive, trivial, or negative. Unclear results are reported if the observed confidence interval overlaps both positive and negative values. The probability of the effect was evaluated according to the following scale: <0.5%, most unlikely; 0.5-5%, very unlikely; 5-25%, unlikely; 25-75%, possibly; 75-95%, likely; 95–99.5%, very likely; >99.5%, most likely (Hopkins et al.,

2009).

17 CHAPTER FOUR

Results

Time Trial Performance and Blood Lactate Response

There was a significant group x time interaction for best TT performance (i.e. fastest time to completion) (p=0.02) (Figure 3). CON demonstrated no change in TT performance from pre- to post-treatment; however, KET improved TT performance as reflected by a 5.8±8.9% decrease in best time-to-completion from pre- to post-treatment

(p=0.02, 95%CI= 2.2, 25.2). When controlling for pre-treatment best TT performance,

KET showed a 19.6-second faster TT than CON at post-treatment (p=0.03, 95% CI= -

36.9-2.3).

When examining the average TT performance across the 2 consecutive trials, there was a significant group x time interaction (p=0.04) (Figure 4). CON showed no change in average time-to-completion while KET demonstrated a significant 3.7% decrease in average time-to-completion from pre- to post-treatment (p=0.04, 95% CI=

0.40-17.2). When controlling for pre-treatment average TT performance, KET demonstrated a 12.1-second faster TT than CON at post-treatment (p=0.04, 95%CI= -

24.4-0.2). There was no significant sex by group by time interaction for either best or average TT performance.

18

Figure 3. Best Time-to-Completion at Pre- and Post-Treatment for Control and Ketone Groups. Values are mean ± SD. * Significant group x time interaction (p=0.02) and significantly faster time-to- completion compared to pre-treatment (p=0.02). ^ Faster best time-to-completion than Control when controlling for pre-treatment measures (p=0.03)

Figure 4. Average (avg.) Time-to-Completion at Pre- and Post-Treatment for Control and Ketone Groups. Values are mean ± SD. * Significant group x time interaction (p=0.04) and significantly faster avg. time-to- completion compared to pre-treatment (p=0.04). ^ Faster avg. time-to-completion than Control when controlling for pre-treatment measures (p=0.04)

19 Both groups demonstrated a significant increase in blood lactate during the TT at pre-treatment (CON= +11.7±4.5 mmol/L vs. KET= +10.4±4.1 mmol/L) (p<0.001) and at post-treatment (CON= +13.6±3.9 mmol/L vs. KET= +11.1±4.9 mmol/L) (p<0.001).

These blood lactate responses did not change from pre- to post-treatment in either group.

There was no group by time interaction on TT blood lactate response (Figure 5). When controlling for pre-treatment blood lactate response, there was no between-group difference in post-treatment blood lactate response. Also, when examining the interaction of group x time on post-TT blood lactate levels with pre-TT lactate measures as covariates, there was no significant interaction. There was no significant group by sex by time interaction on the TT lactate response.

Figure 5. Change in Blood Lactate during TT at Pre- and Post-Treatment for Control and Ketone Groups. Values are mean±SD.

Magnitude-Based Qualitative Inference Analysis

Table 2 presents the outcomes for the magnitude-based inference analysis on the effect of treatment on the pre- to post-treatment change (∆) in best and average TT time-

20 to-completion and TT lactate response. Analysis showed that ketone supplementation is very likely beneficial and likely beneficial in comparison to a placebo with regards to best and average TT performance, respectively. There were unclear inferences for the TT lactate response.

Table 2. Mean values for Pre- to Post-Treatment Change (∆) in Best and Average Time Trial (TT) Time-to-Completion and TT Lactate Response for Ketone (KET) and Control (CON) groups with mean difference, p-values, and qualitative inferences (QI) for effect magnitude.

QI for effect KET CON Mean Variable p-value magnitude (n=10) (n=9) Difference (mean diff ± 90% CL) ∆ Best TT Time to Very Likely Beneficial -5.8 2.4 8.2 0.02 Completion (%) (8.2±2.1) ∆ Average TT Time to Likely Beneficial -3.7 1.0 4.7 0.04 Completion (%) (4.7±5.3) ∆ TT Lactate Unclear Inference 11.6 20.6 9.0 0.65 Response (%) (9.0±35.2)

21 CHAPTER FIVE

Discussion

The overall objective of this study was to determine the effects of one-week exogenous ketone salt supplementation on short-distance running TT performance in male and female endurance-trained subjects. It was hypothesized that following one week of ketone salt supplementation, 800m running TT performance and fatigue resistance across consecutive TTs would improve in comparison to a placebo control treatment. The findings of the present investigation failed to reject our hypothesis. In summary of the current findings, one week of exogenous ketone supplementation resulted in faster 800m

TT performance compared to baseline while the placebo treatment demonstrated no time- dependent effects. In addition, the average time-to-completion across consecutive 800m

TT improved from pre- to post-treatment in the KET group while the CON group indicated no changes over the treatment period. These outcomes for average TT performance may be indicative of improved fatigue resistance following a relatively short-term period of ketone supplementation. Furthermore, the observed effect of ketone supplementation on TT performance does not appear to be mediated by alterations to carbohydrate metabolism or a shift in energy substrate (i.e. fuel) utilization based on outcomes for exercise blood lactate responses. Overall, the present results support the efficacy of a relatively short-term (i.e. 8-day) ketone salt supplementation in aiding short- distance (i.e. 800m) running performance in trained individuals corroborating the outcomes of prior investigations.

In agreement to the current findings, Cox et al. (2016) reported an approximate

2% improvement in maximum distance covered during a fixed-time (30 min) cycle-for-

22 maximum-distance trial following ingestion of a carbohydrate drink combined with exogenous ketone esters using a relative dosing scheme of 573mg/kg bodyweight in comparison to a carbohydrate drink depleted of ketones. Furthermore, Clarke and Cox

(2015) observed similar improvements of maximum distance covered during a 30-minute rowing trial after ingestion of a ketone ester beverage composed of 45% ,

30% ketone esters, 20% protein and 5% fat. Notably, the major differences between the present study and both Cox et al. (2016) and Clarke and Cox (2015) are the dosing schemes, the exogenous ketone form, and the nature of the exercise trial used to examine effects on performance.

First, these prior investigations utilized a dosing scheme individualized to the subject per normalization to bodyweight (573mg/kg bodyweight) and incorporated a ketone ester test supplement. Alternatively, the present study demonstrated the ergogenic value of a fixed ketone dosing strategy of a beta-hydroxybutyrate (ßHB) salt supplement of 11.7g per serving (for at least 8 days) thus highlighting the benefits of both fixed and relative dosing as well as salt and ester forms of exogenous ketones. Certainly, an investigation testing the interaction of dosing strategy and exogenous ketone on some physiological and/or performance-based measure would provide further practical insight.

Accordingly, in a prior investigation, subjects consumed several fixed doses of exogenous ketones (~12 or ~24g) as either a ketone ester (R)-3-hydroxybutyl (R)-3- hydroxybutyrate) or ketone salt (sodium plus potassium ketone) and monitored changes in blood ßHB concentrations. The results showed that both supplemental ketone forms

(within a beverage) transiently raised blood ßHB and induced a state of ketosis (Stubbs et al., 2017) suggesting the effectiveness by which either ketone forms raise blood ketone

23 levels, a fundamental precursor to any ketone-mediated performance benefits.

Furthermore, Clarke and Cox (2015) suggested that the optimal dosing strategy to maintain or improve muscular power output is a single daily dose of 100mg/kg of bodyweight of an exogenous ketone preferably in the form of an ester or (R)-3- hydroxybutyrate-R-1,3-butanediol monoester. However, the current data further begs the question of whether a fixed daily dosing of ketone salts may be a more practical approach, especially considering the cost effectiveness of salt ketone forms. Collectively, these findings may justify further scientific scrutiny as it relates to differences in ergogenic properties between exogenous ketone salt and ester forms as well as relative vs. fixed dosing schemes.

Another notable distinction between the aforementioned prior investigations and the present study is the nature of the exercise trial. Although both Cox et al. (2016) and

Clarke and Cox (2015) demonstrated an approximate 2% improvement in performance measures, aspects of their study design lack relevance and consistency to the real-life conditions of sport competition, especially in cycling, running, or rowing. Both investigations utilized a fixed-time performance test in which subjects were instructed to cover as much distance as possible within a 30-min timeframe. Authors referred to these trials as “time trials” despite no measure of time was implemented (only distance covered). The practical issue here is that running, cycling, and rowing performance, at least in competition, is based on how fast one completes a fixed distance as opposed how much distance one can achieve within a fixed timeframe. Thus, we examined the effects of ketone supplementation using an 800m running time trial in a control laboratory using a motor-less, self-propelled treadmill to better reflect conditions under which athletes

24 would perform a short-distance track competition. This methodological feature adds novelty to our investigation as no studies, at least to our knowledge, have implemented such laboratory-based TT protocol.

In line with our attempt to implement a test protocol more related to real-life competition scenarios, a relatively recent investigation by Leckey et al. (2017) incorporated a very comprehensive study design in which subjects, who were professional cyclists, were tested under conditions of elite professional cycling. This study included ingestion of pre-race carbohydrate-rich meal, a sophisticated warm-up protocol, inclusion of world-class competitive cyclists and laboratory-based simulation of a real-life course. Interestingly, their results oppose the conclusions drawn from the current study as well as Cox et al. (2016) and Clarke and Cox (2015), however some key discrepancies in the ketone treatment and methodologies may explain these divergent performance outcomes. Leckey and colleagues investigated the effects of pre-TT ingestion of a ketone diester (1,3-butanediol acetoacetate diester) on a 31-km laboratory- based cycling TT. Their results demonstrated an impairment of TT performance by approximately 58 seconds following ketone diester supplementation which again challenges the outcomes of prior investigations including the present study (Clarke &

Cox, 2015; Cox et al., 2016). When examining the blood ketone levels of ketone- supplemented subjects from Leckey et al. (2017), plasma ßHB levels were moderately elevated from baseline yet this change was to a lesser degree than what was reported previously (Clarke & Cox, 2015; Cox et al., 2016). Leckey et al. (2017) showed a modest increase in blood ketone levels to 0.3-0.4mmol/L while Cox et al. (2016) showed increased blood ßHB concentrations of roughly 2-4mmol/L despite similar dosages of

25 slightly different ketone forms between studies. Egan and D’Agostino (2016a) proposed an “optimal” range of 1-3mmol/L for performance benefits and may explain the discrepancies in performance outcomes between the two studies. It must be noted, however, that Leckey et al. (2017) reported subjects to have gastrointestinal discomfort following their ketone diester treatment which may have contributed to the impaired TT performance. As for the current investigation, it remains unknown whether the observed performance benefits following ketone supplementation accompanied nutritional ketosis and blood ketone levels within the purported “optimal” range. Therefore, it cannot be fully ascertained whether ketosis was indeed the linking mechanism between ketone supplementation and the ensuing ergogenic response.

In efforts to elucidate mechanisms of action related to fuel utilization shift with ketone supplementation, we assessed the blood lactate response from pre- to post-TT. As expected, both groups demonstrated an acute increase in blood lactate concentrations during the TT both before and after treatment. However, ketone supplementation did not mitigate this exercise blood lactate response, thereby indicating a lack of shift in glycolytic activity. Thus, the performance benefits displayed by KET cannot be explained by a shift in substrate utilization or reliance in anaerobic energy transfer, at least from the present study. These findings, however, remain inconsistent with prior investigations

(Cox et al., 2016; Leckey, Ross, Quod, Hawley, & Burke, 2017). The findings presented by Cox et al (2016) reported blood lactate concentrations ~1.5-2mmol/L lower after a 60 min 75% Wmax and 30-min exercise trial for subjects consuming a ketone ester CHO beverage compared to a CHO beverage alone. Whereas similar results were reported by

Leckey et al. (2017) who observed lower post-TT blood lactate concentrations following

26 ketone treatment compared to a placebo (8.6 ± 3.2 vs. 13.1 ± 4.3 mmol/L). However, the performance trials used by Cox et al. (2016) and Leckey et al. (2017) were longer in duration and naturally lower in intensity compared to the present study which implemented a short-distance running trial of 800m. Also, both studies demonstrated exercise lactate levels of ~2-10mmol/L which falls below the average post-TT lactate levels found within the present investigation (~14.5 mmol/L). This suggests that the current performance trial involved greater relative intensities considering the positive linear relationship between blood lactate and exercise intensity (Coggan, Raguso,

Williams, Sidossis, & Gastaldelli, 1995). It can be speculated that our TT protocol involved a relatively higher exercise intensity necessitating elevated intramyocellular glycogen and glucose reliance for energy provision (Balaban, 1990; Coggan et al., 1995).

Perhaps, exogenous ketone intake may simply provide additional energy substrate without shifting fuel utilization away from carbohydrate sources especially during higher workloads or relative intensities. This question warrants further investigation relating to the interaction between exogenous ketone supplementation, intensity of subsequent exercise performance, and energy substrate utilization.

Overall, the results of the present study demonstrated that one-week ketone salt supplementation provides a slight increase on running TT performance on a non- motorized treadmill. Thus, athletes may gain a small performance advantage from the use of a fixed (11.8g of sodium βHB) and relatively short-term (~1 week) ketone salt supplementation regimen. Although, further research is warranted to determine the optimal supplementation protocol especially regarding the interaction of dosing strategies and exogenous ketone forms. Additionally, the efficacy of ketone supplementation should

27 be further examined longitudinally as it relates to the degree by which long-term training adaptations may be influenced. Also, less is known regarding the ergogenic value of exogenous ketones for general fitness (non-competitive or athletic) populations, thus, future investigations in various populations may be warranted. In conclusion, our results agree with prior investigations supporting the efficacy of exogenous ketones to increase sport performance.

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