1 Effects of a Ketone-Caffeine Supplement on Cycling And

Home , Alcoholic ketoacidosis, Exogenous ketone

Effects of A Ketone-Caffeine Supplement On Cycling and Cognitive Performance in

Chronic Keto-Adapted Participants

THESIS

Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in

the Graduate School of The Ohio State University

Madison Lee Bowling

Graduate Program in Kinesiology

The Ohio State University

2018

Thesis Committee

Dr. Jeff Volek

Dr. William Kraemer

Dr. Carl Maresh 1

Copyrighted by

Madison Lee Bowling

2018

Abstract

As research begins to broaden our understanding of the effects of low carbohydrate, high fat ketogenic diets to different populations, it is crucial to utilize evidence associated with the metabolic and physiological adaptation of chronic implementation. Specific populations are finding that nutritional ketosis may prove advantageous to athletic or cognitive performance. Nutritional ketosis may be identified by an elevated plasma ketone concentration within the blood range 0.5 to 5 mmol/L that results from a chronic implementation of a ketogenic diet. Recently, science shows that ketones contribute to a vast range of therapeutic and performance benefits associated with nutritional ketosis, as a result, exogenous ketone supplements have become commercially available which have proven to induce acute nutritional ketosis without restriction of carbohydrate intake. We previously showed that a supplement containing ketone salts and caffeine significantly increased performance in a non-keto adapted population. To date, there are no reports of whether ketone supplements have an ergogenic effect in an already keto-adapted population. The primary purpose of this study was to determine the performance and metabolic effects of a supplement containing ketone salts and caffeine in a group of people habituated to a ketogenic diet. Twelve habitually ketogenic,

recreationally trained individuals (3 female, 9 male: mean + SD age, 36.1 + 7.5 years; weight, 82.2 + 7.1 kilograms; height, 177.7 + 8.5 cm; VO2max 40.3 + 10.5 ml/kg/min). participated in two experimental sessions in a randomized and balanced order. Subjects consumed either a ketone-salt/caffeine supplement containing 7.2 BHB and 96.2g caffeine or water (control condition) 15 min prior to performing a staged cycle ergometer time to exhaustion test followed immediately by a 30 sec Wingate test. Symbol digit modality tests were administered at baseline, immediately post-exercise and 30-/60-min post-exercise. Blood ketone concentrations were significantly increased peaking 15 min after ingestion by more than 2-fold and staying elevated throughout 60 min recovery.

Compared to the water trial, ingestion of the ketone-caffeine supplement significantly increased time to exhaustion (9.8%; P = 0.003) and increased peak VO2 during exercise

(12%; P = 0.03). There were no significant differences between conditions in peak power output, average power output, cognitive performances, or blood glucose responses. These results indicate that ingestion of a moderate dose of ketone salts and caffeine prior to exercise significantly increases the magnitude of ketosis and improves high-intensity exercise performance in keto-adapted individuals.

iii

Acknowledgments

I would first like to begin with thanking Dr. Volek, my mentor, advisor, and inspiration throughout my research as a graduate student. Not only do I feel my knowledge and professional experience has increased under your aid, but my passion for the research we have been devoted to. Through your guidance, I learned both perseverance and patience for the hands we are dealt, both personally and professionally.

I am looking forward to continuing my doctoral education under your mentorship.

To Dr. William Kraemer and Dr. Carl Maresh, thank you for serving on my committee. I have learned many things from the both of you, whether you meant to teach them to me or not. Through shadowing the both of you, I was able to observe my own idea and questions through other perspectives, bringing me closer to finding my “nitch”.

Team Volek- Jay, Parker, Rich, Emily, Teryn, Anna, Ryan, Vin- I learned an incredible amount in the first 2 years of my graduate career from all of you. Whether it be

“baptism meet fire” or a Vin-worthy blood processing manual on a Saturday morning. I

To my boys, Ryan and Ryder- I can’t possible explain how much I needed you both to be by my side as I wrote this chapter of my life. Thank you both for being more than I ever thought I needed. Whether it was the copious amounts of coffee you kept coming, the unceasing support, or total family homework time- I can’t thank you enough for being by my side, boys. I love you. #TeamKackley

To Mom, Morgan and Liberty- this road has seemed almost impossible hasn’t it?

Here we are. I may have doubted when nothing seemed possible but you guys didn’t doubt me for a second. Thank you for the love, the overwhelming peace, and all the prayers. I love you all so much. I promised I’d make you proud.

To my very best friends; Chey, Mak, Char, Emily, Allie, Claire, Justin, Ant,

Mann, Hollywood, Sabrina, Marie, Jenn, TJ, Annette, Kailee. I love you all. Thank you for listening to me vent, pushing me forward, bringing me back to reality, and of course- all the nights I needed to breathe. I love you all for every part of your soul. I would not be here, where I am, without any of you.

Vita

2010…………………………………….... Northridge High School

2014……………………………………… B.A. Exercise Science, Otterbein University

2016 to present…………………………… Graduate Associate, Department of

Kinesiology, The Ohio State University

Fields of Study

Major Field: Kinesiology

Table of Contents

Abstract ...... ii

Acknowledgments ...... iv

Vita ...... vi

List of Figures ...... x

List of Tables ...... xi

Chapter 1. Introduction ...... 1

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

2.1 Ketone Metabolism/Physiology ...... 5

Metabolic Effects of Ketones ...... 7

Non-Metabolic Effects of Ketones ...... 9

2.2 Well-Formulated Ketogenic Diet ...... 10

2.3 Ketone Supplementation ...... 12

vii

Chapter 3: Methods ...... 15

Experimental Approach ...... 15

Subjects ...... 16

Supplement ...... 17

Testing Preparation ...... 19

Baseline Testing ...... 19

Full Test Day: T1 & T2 ...... 21

Blood Processing and Analysis ...... 23

Statistics ...... 23

Chapter 4: Results ...... 25

GI Distress ...... 25

Ketone and Glucose Levels ...... 25

Time to Exhaustion ...... 26

Peak V02 ...... 26

Wingate Power ...... 26

Cognitive Testing ...... 26

Rate of Perceived Exertion ...... 27

Respiratory Exchange Ratio ...... 27 viii

Lactate ...... 27

Glycerol ...... 27

Chapter 5: Discussion ...... 28

Conclusion ...... 34

Bibliography ...... 35

Appendix A: Figures ...... 45

Appendix B: Tables ...... 48

List of Figures

Figure 1: Study timeline…………………………………………………………………16

Figure 2: Supplement nutrition label…………………………………………………….18

Figure 3: Nutrition Analysis……………………………………………………………..18

Figure 4: Test day Timeline………………………………………...……………………24

Figure 5: Capillary Blood Beta-Hydroxybutyrate responses……………...……………..45

Figure 6: Capillary Blood Glucose responses……………………………………………46

Figure 7: Individual Responses in Time to Exhaustion………………………………….47

List of Tables

Table 1: Nutrient Comparison..……………….…………………………………………19

Table 2: Participant Characteristics...………………………...………………………….48

Table 3: Peak beta-Hydroxybutyrate and TTE Response ……..…….…………………..49

Table 4: Wingate Power Output …….………………………..…………………………50

Table 5: Respiratory Exchange Ratio……………………...……………...……………..51

Chapter 1. Introduction

Adaption to a ketogenic diet and its physiological benefits are becoming more apparent to humans. Historically, increased ketones and utilization in metabolic pathways were paralleled with the harsh scrutiny of starvation, prolonged periods of food deprivation or pathologic conditions like diabetic ketoacidosis, alcoholic ketoacidosis, and salicylate poisoning (Laffel, 1999). Today, research is moving forward to detangle the misunderstanding of elevated ketones and low-carbohydrate dietary interventions.

Until resent literature, the idea of starvation or carbohydrate restriction, when dietary carbohydrate intake is restricted to less than ~40grams/day which innately accelerates the production of ketones in order to maintain energy availability, has been widely unaccepted. Within several days on a “ketogenic” or high-fat/low carbohydrate diet, circulating ketones increase from <0.2 to 0.5-4.0 mmol/L, a range referred to as

‘nutritional ketosis’. Several weeks of nutritional ketosis will lead to metabolic changes that allow inner-organ fuel supply to be primarily fulfilled by fatty acids and ketones in situationally low- carbohydrate availability. Mechanistically, ketones have been advocated as potent signaling molecules that positively affect gene expression and cellular function (Newman & Verdin, 2014). Ketone metabolism has recently been

studied in application to athletes or military personnel who need to preform both physically and cognitively under stressful conditions due to the decrease in glucose availability with prolonged periods of stress (Brownlow, Jung, Moore, Bechmann, &

Jankord, 2017; Mcnay, Fries, & Gold, 1999; Yuen et al., 2012). Ketogenic diets have therapeutic effects on insulin resistant conditions and neurological disorders such as type

2 diabetes, metabolic syndrome and epilepsy (G. Cahill, 2006; Keon et al., 1995; L.

Veech, Britton Chance, Yoshihiro, Chance, Kashiwaya, Lardy, & Cahill, 2001; Veech,

2004). Ketogenic research has also begun a breakthrough in both physical and mental performance. Athletes and military personnel often need to perform physically and cognitively under stressful conditions. Research shows availability of glucose, a typical fuel source for these demographics, decreases (Mcnay et al., 1999; Yu, Tompkins, Ryan,

& Young, 1999) . The number of athletes that have switched to a ketogenic diet have grown due to recent discoveries. Some of these athletes have gone on to win races, setting records; both personal and national(Volek et al., 2016).

Whereas sustained nutritional ketosis results from the habitual restriction in dietary carbohydrate, it is now possible to elevate ketones acutely by ingesting ketone supplements with or without carbohydrate restriction. Historically, research has investigated the safety of ketone esters (Clarke, 2013) and the ability to improve physical performance (Cox et al., 2016) due to ketones efficiency at producing ATP for energy (G. Cahill, 2006; Keon et al., 1995; L. Veech, Britton Chance, Yoshihiro et al.,

2001; Sato et al., 1995). Ketones are a primary energy substrate that can cross that blood brain barrier to fuel brain energy requirements when glucose availability is limited (G.

Cahill, 2006; Keon et al., 1995; Laffel, 1999). Which has made manufacturing of ketone supplements to be advantageous to the athletic population. The safety of these products has been evaluated and not associated with significant side effects (Clarke, 2013;

Hashim & VanItallie, 2014). Recent research has studied trials administering exogenous ketones orally to select populations. Leckey et al. reported consumption of a ketone diester impairs 31km cycling trial performance but the effects of this impairment may not be associated with exogenous ketone intake due to questionable formulation and dosing.

The study monitored both a supplement group of 1-3-butanediol-acetoacetate, not beta- hydroxybutyrate, and a control finding a decrease in performance of the supplemental group. However, it is unclear as to what caused the decrease in performance considering multiple complications; 3g/kg carbohydrate pre-exercise, 27g CHO Powerbar, 200mL diet cola, or possibly the ~8.5oz of Gatorade, mid-test may add to a number of confounding variables associated with performance(Leckey, Ross, Quod, Hawley, &

Burke, 2017). Other studies have utilized exogenous ketone products with miscalculation of the product formulation (O’Malley, Myette-Cote, Durrer, & Little, 2017). Upon further investigation of ketone esters and ketone salts, it is necessary to understand that the formulation of both contains different compounds of the ketone isoforms. While D-BHB and L-BHB can both increase blood level nutritional ketosis, a formulation with high L-

BHB may do little on athletic performance. (Stubbs et al., 2017) 3

Short et al. (in review) studied the effects of a ketone salt and caffeine supplement on performance in 12 keto-naïve, recreationally trained individuals. This cross- cross over study included 2 identical exercise trials; Time to Exhaustion trial of 95% max followed by an all-out 30sec Wingate, with a pre-exercise ingestion once of supplement and once of water control condition. Results showed an 8.3% increase in Time to Exhaustion and a

4.4% increase in average power during the Wingate protocol. Another study with a similar testing protocol reported an ~2.5% average power (Rodger, Plews, Laursen, &

Driller, 2017). However, with the several studies that examine the effects of exogenous ketones, none were conducted in an already keto-adapted cohort. Therefore, the primary purpose of this study was examine the performance and metabolic effects of acute ingestion of a supplement containing ketone salts and caffeine. Manufacturers often combine ketones with a caffeine product in order to mildly increase lipolysis, decrease perceived exertion, and increase performance (K. Wang, McCarter, Wright, Beverly, &

Ramirez-Mitchell, 1991). We hypothesized that keto-adapted individuals would show elevated ketones and fat oxidation, and experience a similar ergogenic and metabolic effect to acute ingestion of the ketone-caffeine supplement.

Chapter 2. Literature Review

Historically, exogenous ketone supplementation had only exclusively been studied in clinical settings where the mode of delivery into the body was intravenous infusion (Dye &

Chidsey, 1939; Wick & Drury, 1941). As the research and interest in the benefits of a ketogenic diet increases, it is appropriate to investigate the possible outcomes of the effect of exogenous ketone supplementation on performance and metabolism. As more exogenous ketone products

(such as Pruvit’s KETO//OS MAX CHARGED) become commercially available, it is also necessary to invest in the research of its efficacy on both mixed or high carbohydrate diet and habitually low carbohydrate, high fat fueled populations. While popularity of a ketogenic diet and keto-adaptation has had quite an impact of performance, exogenous ketones have also been studied in parallel to performance enhancement and metabolic differentiations.

2.1 Ketone Metabolism/Physiology

Current research has allowed for the formulation of exogenous ketones to be administered orally. The ketones can be consumed and utilized when bound to two forms; esters and salts. Even as salts have become commercially more popular, both have been shown to be successful at elevation blood plasma ketone levels to achieve acute Ketosis(Balasse & Neef,

1978; Clarke, 2013; Cox et al., 2016; Stubbs et al., 2017). The safety of ingestion of exogenous 5

ketone salts and ester supplementation have been demonstrated with minimal side effects

(Clarke, 2013; Shivva et al., 2016; Veech, 2004). Some individuals may experience mild adverse symptoms upon ingestion including: flatulence, vomiting, nausea, diarrhea, constipation, abdominal distension. However, the side effects are unable to be attributed to the exogenous ketones independently due to the volume of added consumption (fluid or latter) during recently reported protocols (Clarke, 2013; Leckey et al., 2017).

Ketone bodies are the chemicals that are produced in the liver when fatty acids are broken down in excess. Breakdown into these compounds is called ketolysis. The three ketone bodies formed are: acetone, acetoacetate and b-Hydroxybutyrate. These are derived from an important coenzyme, acetyl-CoA, notable for its role in lipid oxidation mainly in the mitochondrial matrix of the livers cells when carbohydrate fuel sources are low. This breakdown allows for energy to be created via the breaking down of fatty acids and that energy to be utilized in peripheral tissues. With high levels of ketones, glucose utilization can be migrated, where ketones can then be used as a primary fuel, especially in the brain (Lamanna et al., 2009;

Ruderman, Ross, Berger, & Goodman, 1974). Mechanistically, ketones can be used in the TCA cycle at the level of citrate, which will bypass glycolysis, downregulating glycolytic rate increasing the ability for fatty acids to be broken down and utilized for energy.

Ketone bodies such as b- Hydroxybutyrate (BHB) and Acetoacetate (AcAc) can increase with induction of ketosis, usually attainable through a chronic ketogenic diet (L. Veech, Britton

Chance, Yoshihiro et al., 2001; Veech, 2004) Ketone plasma levels achieved via exogenous

supplementation in previous studies have ranged from <0.5 mmol/L at baseline measurements to around 3.5 mmol/L at some of the high level ingestion of ketones in a non-keto-adapted (keto- naïve) state(Clarke, 2013; Cox et al., 2016). The range of blood plasma ketones is of importance as ketone levels are <0.5 mmol/L if keto-naïve, 0.5-4mmol/L during a chronic consumption of a well formulated ketogenic diet and between 5-8mmol/L after prolonged starvation (G. Cahill,

2006; L. Veech, Britton Chance, Yoshihiro et al., 2001). All these levels are physiologically safe in most population. The urgency to check further altercations would have to reach measurements of blood plasma ketone levels of around 25mmol/L generally associated with diabetic ketoacidosis, which can become potentially harmful(Veech, 2004). The ability to reap the benefits of keto-adaptation without the adherence to a well-formulated ketogenic diet is a concept the world has increasingly found intriguing. However, as exogenous ketones are preferentially studied in those who are keto-naïve for acute ketosis, it proposes dose response questions for those who are already on a chronic fat fueled metabolic state. With those who are keto-adapted we may see even further benefits of adaptation if also supplemented with exogenous ketones. Ketone consumption may work synergistically with the keto-adapted pleotropic effects they already experience and advance further performance both mentally and physically.

Metabolic Effects of Ketones

Ketosis is the metabolic state at which the body burns stored fat when glucose storages are low to utilize for energy. In opposition of the association to ketoacidosis and a low-

carbohydrate diet, this state or nutritional ketosis is harmless for the body (Manninen, 2004).

Historically, general populations have been told that the primary source for energy in our blood is sugar. Ketosis allows for blood plasma levels of ketones to increase from an average

<0.5mmol/L to >0.5mmol/L. This increases energy derived from fat fuel sources and upregulates fatty acid gluconeogenesis. As research investigates energy substrates further, we are unable to deduce a clear requirement for carbohydrates even for the brain. Furthermore, it has been studies that 60% of the brains energy can be derived from ketones (G. Cahill, 2006) without altering oxygen consumption (Ruderman et al., 1974).

Fatty acid metabolism is a notable metabolic factor when a person’s body performs in a ketogenic state. The beta-oxidation pathway has the ability to oxidize Beta- hydroxybutyrate and provide energy by conversion to acetyl-CoA then entering the TCA cycle for ATP production

(Laffel, 1999). Ketolysis has the potential to generate energy quicker than glycolysis due to fewer steps needing in order to the production of acetyl-CoA to occur and enter the TCA cycle.

Not only does this process adhere to speed, but efficiency as well due to the ability of ketones to produce more energy per unit of oxygen (G. F. J. Cahill & Veech, 2003; Sato et al., 1995).

During the state of Ketosis energy can be created quickly and efficiently which would initiate interest in anaerobic performance in a ketogenic state especially in high stress situations where it is found that glucose availability will decrease as the task, both mental and physical, becomes higher in demand (Mcnay et al., 1999; Yuen et al., 2012). As increase in stress intensity has a substantial effect on the availability of glucose for energy this has the same relationship with

prolonging bouts of stress. Recently, endurance athletes have opened the doors for assessing the pleotropic benefits of a ketogenic state to negate this drop off. Research has concluded that these benefits have aided in performance and at the very least matching what can be done in a higher carbohydrate diet (Volek et al., 2016).

Non-Metabolic Effects of Ketones

One of the most notable non-metabolic effect of ketosis is the increase of b-

Hydroxybutyrate (BHB) and acetoacetate (AcAc) (Maalouf, Sullivan, Davis, Kim, & Rho, 2007) and its effects on reactive oxygen species (ROS) (Shimazu et al., 2013). Ketones can increase hydraulic efficiency in the heart through mitochondrial ATP production by ketone metabolism.

Due to mechanistic efficiency of oxidative phosphorylation to derive ATP from the electron transport chain, the breakdown of ketone bodies, AcAc and BHB, adds to the energy output without increasing mitochondrial production of reactive oxygen species but increasing NADH oxidation (L. Veech, Britton Chance, Yoshihiro et al., 2001; Maalouf et al., 2007), thus would lead to improved recovery and increase longevity. This eludes to ketones protective effect on brain in such ways as preventing death of hippocampal neurons and reduction to brain injury in glycolysis inhibition (Keller et al., 2005; Maalouf et al., 2007). Ketones have also benefited in people with glioma as an adjuvant therapeutic prescription (Stafford et al., 2010).

Cellular metabolites such as acetyl-coenzyme A (acetyl-CoA) and nicotinamide adenine dinucleotide ( NAD+) are heavily impacted by increased BHB abundance. Both metabolites are correlated with the inhibition of histone deacetylase (HDAC) (Shimazu et al., 2013). The 9

inhibition of HDAC has been associates with genomic functions such as DNA damage repair and cell cycle regulation. Treatment of cells with BHB will increase histone acetylation by FOXO transcription factors, which are at the interface of cellular processes and gene expression allowing for regulation of apoptosis, cell-cycle progression, and stress resistance (Carter &

Brunet, 2007; Shimazu et al., 2013). High fat caloric intake and a ketogenic dietary intervention has also demonstrated significant reduction of the expression of lipid peroxidation factors which may be contributed to the increase in mitochondrial superoxide dismutase (SOD) which may contribute to the protection of oxidative damage (X. Wang et al., 2017). Little information is available to the public to show whether consumption of exogenous ketone supplementation in acute ketosis will mitigate ROS post- exercise.

2.2 Well-Formulated Ketogenic Diet

Historically, ketogenic adaptation is associated with starvation as the influx of plasma ketone concentration is affiliated with diabetic ketoacidosis. The ketogenic diet (KD) has recently been coined as a metabolic state to which a body utilized fat stores as a primary energy source. During the breakdown of adipose tissue in the liver, ketones are made readily available in order for energy. A well formulated ketogenic diet contains between 3-20% carbohydrates and between 10-30% protein, with the rest of the caloric intake derived from fat. This carbohydrate restriction is an idea that has been adopted by many ancient cultures and is circumstantially becoming more applicable to a modernistic approach to wellness. KD results in a glycogen sparing phenotype and a shift to reliance on lipid-based fuels.

A ketogenic diet may have large role in control of hunger and satiety when athletes deal with weight regulation. It is popular to associate a ketogenic diet with starvation and caloric restriction but that is not an applicable view of this complex relationship. Ketone bodies, such as

BHB and AcAc are able to increase AMP- activated protein kinase (AMPK) during orexigenic properties (Harney et al., 2017; Paoli, Bosco, Camporesi, & Mangar, 2015). Even though an athlete can regulate food intake by will, the signaling within the central nervous system can intrinsically mediate food intake and energy expenditure. Habitually, the brain can detect alterations in energy storages and trigger metabolic and behavioral responses to maintain energy balance. According to “glucostatic hypothesis” a decrease in blood glucose in a fasted state can initiate the orexigenic necessity for food consumption, however it is the adipose tissue in which signals are released into the brain to control for feeding and weight maintenance (Kennedy,

1953; Rexford S.A., 2009). AMPK activation is upregulated in a ketogenic state allowing for the replenishing of cellular energy by ways of fatty acid oxidation which negatively regulating ATP- consuming processes such as gluconeogenesis. This pathway may spare energy for athletes during the process of consumption to save for activity.

KD may have a performance benefit on athletes in opposition of the earlier literature suggestion for high carbohydrate consumption. Most athletic enthusiasts found it necessary for peak performance outcomes (Helge, 2017), thus accentuating the dogma associated with short- term high carbohydrate interventions for endurance athletes. Research comparing short-term carbohydrate intervention vs. short-term fat intervention solidified athletic stand against low

carbohydrate diets. Recently, athletes have refuted this dichotomy by applying carbohydrate restriction to their athletic performance and finding reprieve (VOLEK). Science has now seen performance enhancement to some athletes due to nutritional ketosis from a chronic carbohydrate restriction possibly finding a more competitive phenotype. Due to the increased utilization of lipids for fuel and decrease in insulin flux from scarce carbohydrate will led to an increase in hepatic production of BHB and AcAc for energy in active muscle and the brain during prolonged periods of stress and decreased glucose availability (Brownlow et al., 2017; G.

Cahill, 2006; Mcnay et al., 1999; Volek et al., 2016). Higher rates of fat oxidation and increase in oxidation of BHB would theoretically delay the utilization of glycogen stores mitigating the

“bonk” that occurs with prolonged endurance activity and would lead less calories being consumed during long sporting events (Volek, Noakes, & Phinney, 2014). Keto-adapted athletes have even demonstrated like glycogen metabolism to high-carbohydrate matched controls (Volek et al., 2016). Even during utilization of KD in weight loss, it was found that elite male gymnast who had decreased weight maintained pre-intervention strength performance (Paoli et al., 2012).

2.3 Ketone Supplementation

As ketones have quickly become more popular, supplement companies have increase production in the products available to allow for the most performance benefit. Ultimately, the sales schematic has led to a rise in the purchase and intake in society making it necessary to validate the possible benefits of these products. Safety and efficacy have already been established (Clarke, 2013; Stubbs et al., 2017) more products are becoming commercially

available. With the promoting factor of these products being to elevate blood concentrations of ketones to achieve an acute state of “nutritional ketosis”. Research is still naïve to the parallel acute ketosis via exogenous ketones and the pleiotropic effects attributed to chronic ketosis via keto-adaptation. Research has already established the ability to improve physical performance via exogenous ketones (Cox et al., 2016) and nutritional ketosis (Cox et al., 2016; Paoli et al.,

2012; Volek et al., 2016, 2014) but has yet to evaluate a dose response of plasma ketone concentration. Ultimately, there does appear to be a difference in the body’s ability to utilize ketones as an energy source depending on concentration. When plasma ketone concentrations are elevated around 1-2mmol/L, the body is able to take up and utilize them up to five-times faster at the onset of exercise (Balasse & Neef, 1978; Fery & Balasse, 1985; Féry & Balasse, 1988).

However, if ketones levels are too high, the clearance rate may not be impacted by onset (Féry &

Balasse, 1988). Consequentially, when the ketone levels are higher, the body has a harder time in clearance to bring levels back down to baseline assuming higher ketones induce slower uptake

(Clarke, 2013; Fery & Balasse, 1985; Féry & Balasse, 1988). Exogenous ketones may have a longevity that isn’t seen with the fast clearance of glucose for energy, thus initiating the question of whether a person on a chronic KD diet and exogenous ketone supplementation make for the performance enhancing formula the world has been waiting for. Theoretically, this would result in improved mental and physical performance endurance with longer and/or increased power production without being effected by the loss of glucose availability (Cox et al., 2016; Mcnay et al., 1999; Yuen et al., 2012).

Exogenous ketones have proven effective for altering metabolism into an acute state of ketosis. The rise in BHB and AcAc and elevation of ketone plasma concentrations to

>0.5mmol/L in the body are a primary outcome of ingestion of exogenous ketones. The driving qualities of elevated ketones can be marketed in these products are “lasting energy” and “fat burning”. Given the phenotype of ketosis, it is not wrong for companies to suggest. The current study is designed as a two arm, multi-factor overlook on ketone supplementation with mental and physical performance testing. The novel implication is the keto-adapted arm that will allow us to assess appropriate the body’s ability to utilize high level concentration of plasma ketone.

This will allow us to answer appropriate whether too much ketone can inhibit performance or validate our hypothesis concluding heightened performance response and a positive relationship with ketone levels.

Chapter 3: Methods

Experimental Approach

This study was conducted using a crossover control trial design comparing ketone- caffeine supplementation versus a control condition (water). Participant were required to visit the lab on four occasions to complete the consent process, maximal aerobic testing and familiarization session, and two experimental exercise sessions: one where the subject ingested a ketone-caffeine supplement and the other test day replicating the very same testing protocol but with a control water condition. The two experimental sessions were performed in a randomized and balanced order using a number randomization generator. Each exercise test day lasted approximately 2.5 hours with a 2-7 day rest period in tween testing. This schedule was determined upon the subject’s availability to return and the necessary time to recover from the physical exertion of the test. Overall, the study took 1-2 weeks for each participant. The specific test days are listed below:

1. Screening/Complete consent form

2. Baseline Testing – Vo2Max Cycle Test; cognitive test

3. Cycling test & cognitive testing with ketone supplement

4. Cycling test & cognitive testing with control condition (water)

Subjects

12 recreationally trained keto-adapted subjects participated in this study (Table 1) (3 female, 9 male: mean ± SD; age, 36.08 ± 7.5 years; weight, 83.6 ± 9.67 kilograms; height, 177.9

± 7.68 cm; VO2max 40.3 ± 10.47 ml/kg/min). To ensure eligibility, participants completed a medical history questionnaire, physical activity questionnaire and a food frequency questionnaire before signing an informed consent document approved by the institutional review board. The inclusion criteria for this study required participants to be; 18-50 years of age, recreationally trained of which guidelines are explained in ACSM recommendations of at least 150 minutes of moderate activity per week and habitually consuming a ketogenic, low carbohydrate, high fat, moderate protein diet for at least a month. Keto-adaptation eligibility was determined by several measures including, 1) scrutiny of a food frequency questionnaire and food logs for a low carbohydrate content (<50 g/day), 2) a resting exchange ratio <0.75, which is typical in keto- 16

adapted athletes (Phinney, Bistrian, Evans, Gervino, & Blackburn, 1983; Volek et al., 2016;

Zajac et al., 2014) and 3) fasting ketones >0.5 mmol/L. Participants were excluded if the administered questionnaires detected; metabolic or endocrine dysfunction, cardiovascular or respiratory diseases, gastrointestinal disorders, regularly smoking, consuming alcoholic beverages in excess of three drinks per day, epilepsy, chronic headaches or if pregnant.

Participants were asked to discontinue if able any use of probiotics, antibiotics, antifungals and any supplements known to impact exercise performance, antioxidant or inflammatory status.

Supplement

According to the manufacturer, the supplement used in this study consisted of 7.2 grams of beta-hydroxybutyrate in the salt form (sodium, magnesium, and calcium) and 100 mg caffeine

(KETO//OS MAX CHARGED, Pruvit) (Figure 2). One 18.4 g individual serving supplement packet was mixed with 16 oz of water prior to ingestion. During the control condition, subjects consumed an equal volume of water only. Total energy, mineral and caffeine content were validated by an independent laboratory (Covance, City, St) (Table 3). Comparison shows similar nutritional quantification (Table 1).

Figure 2: Supplement Nutrition Label

Figure 3: Nutrient Analysis

Table 1: Nutrient Analysis Comparison

Testing Preparation

Prior to testing, participants completed a 24 hour food record. Participants were asked to replicate the same eating pattern before the second day of testing. Subjects avoided any food and caloric beverages within 8 hours of their scheduled testing time to ensure they arrive at the lab in a post-absorptive state. The participants were specifically advised to avoid any alcohol, caffeine and voluntary physical activity the day immediately prior to testing. Subjects we advised to maintain their current activity level throughout the study, with the exception of the day before testing. At this time, subjects were advised to avoid exhaustive exercise in order to prepare for the following day.

Baseline Testing

Participants arrived at the lab in the morning having fasted for at least eight hours prior to arrival and without participating in any voluntary exercise the day before. Participants were to

bring in a 24hour food diary in order to ensure by macronutrient analysis that participants were still in a state of ketosis. Subjects were advised to pick an easy replicable day of meals to recreate each day prior to testing. Upon arrival they were asked to provide a urine sample to determine specific gravity (Reichert TS 400 clinical refractometer) to ensure acceptable hydration was met (all subjects had a USG <1.025). Female participants completed a urine pregnancy test (Sure-Vue urine hCG strips). Height and weight were assessed (seca 763,

Deutschland) before being familiarized with the symbol digit modality test (SDMT), which was used to measure cognitive function. SDMT is a 90 second test in which there is a key with nine unique shapes each representing a specific number 1-9. The participant was given a series of those shapes and asked to write the corresponding number for each one in order to complete as many as they can as accurately as they can. It was explained that each SDMT would look different each time the test was administered to impede ceiling effect familiarization.

Participants were then familiarized with the equipment and procedures before completing a maximal oxygen consumption (VO2max) test on a stationary bicycle (Lode Corival bicycle ergometer, Groningen, Netherlands) using indirect calorimetry (Parvo Medics TrueOne 2400

Metabolic Measurement System; Sandy, Utah). Participants were fitted with headgear and a mouthpiece attached to a hose (HANS RUDOLPH 2700 Series non-rebreathing; Shawnee,

Kansas) linked with the two-way air chamber (Parvo Medics TrueOne 2400 Metabolic

Measurement System; Sandy, Utah) with a nose-clip to ensure all air is collected from the mouth and not escaping out the nose. The protocol began with a 4 min warm-up period with zero

resistance to allow adjustment of equipment for comfort and proper function while cycling. The staged protocol began at 20W of resistance and increased 20W every 60 sec. The test was completed when the participants voluntarily stopped pedaling, occurring within 18 min.

Measurements of VO2 and VCO2 via breath-by-breath gas exchange was recorded every

15 sec. These values were used to determine oxygen uptake, carbon expiration and respiratory exchange ratio (RER) to calculate carbohydrate and fat oxidation rates. VO2 peak and RER were determined by the highest 15 sec interval value reported. After testing, the participant was given a food log to record what they ate during the 24 hr prior to the upcoming test day and a date for the first test session was scheduled within 7 days and after at least 48 hours to allow for adequate recovery.

Full Test Day: T1 & T2

Subjects arrived to the testing center fasted with their dietary log completed. All participant’s hydration status was measured via urine and female participants completed a urine pregnancy test. A finger stick was conducted with a lancet to obtain capillary concentrations of beta-hydroxybutyrate and glucose at baseline (FreeStyle Optium beta- Ketone Blood beta-

Ketone Test Strips, Abbott Nutrition, Columbus, Ohio) and glucose (ReliOn ULTIMA Blood

Glucose Test Strips, Abbott Nutrition, Columbus, Ohio) using a handheld meter (Precision Xtra

Blood Glucose and Ketone Monitoring System, Abbott Nutrition, Columbus, Ohio). Upon completion, a baseline SDMT test was conducted prior to consuming the supplement or water,

which was ingested within 5 min. Another finger stick was taken 10 and 15 min after beverage consumption. Subjects were fitted with the mouthpiece attached to the metabolic cart. The bike seat was positioned to be exactly the same as it was for the participant at baseline. An aerobic graded exercise protocol to exhaustion was then performed. This protocol was piloted with a test-retest reliability analysis before implementation (Cronbach’s Alpha > 0.7). Testing began with a 4 min warm-up period to allow for participants to get comfortable and ensure all equipment was properly positioned and functioning. Once started, each stage lasted 5 min starting at 65% of their determined VO2max. Each stage increased by 5% of their VO2max until

90% was reached where the resistance was maintained until exhaustion. At the end of each stage a rate of perceived exertion was collected using the 6-20 BORG RPE scale. One finger stick was taken at the end of the third stage to determine changes in ketones and glucose at the predicted mid-point. Exhaustion was defined when the revolutions per minute (RPM) on the bike became less than 60. The first time the participant fell below 60 RPM, a verbal warning was given and the participant had 5 sec to get the RPMs above 60 at which point testing resumed. If the participant was unable to get the RPMs back up within 5 sec or the second time the RPMs fall below 60, the test was ended. After exhaustion was achieved, the mouthpiece and headgear were removed and the participant was immediately moved to a second bike (Monark Ergomedic 894E bicycle ergometer, Vansbro, Sweden) to complete a 30 sec anaerobic Wingate test. The bike seat was adjusted prior to testing having begun and set to same measurements for both test days.

Once completed the participant immediately completed another SDMT test followed by a finger stick and completed a gastrointestinal distress questionnaire that involved a 0-10 point scale. The 22

participant was then seated for 60 min to recover. Finger sticks were taken every 10 min throughout the recovery with a third and fourth SDMT completed at 30- and 60-min recovery.

Upon completion of test session one, participants were given a copy of their completed food log to replicate the day before their scheduled second test day in which the same protocol was followed with the alternative beverage being consumed.

Blood Processing and Analysis

A butterfly needle was inserted into the antecubital vein of the subject. Approximately 12 mL of blood was drawn at each time point into serum or EDTA tubes. Venous blood was centrifuged to obtain plasma or serum, and aliquoted into appropriately labeled microcentrifuge tubes. Samples reserved for lactate measurement were deprotonated using meta-phosphoric acid according to assay kit recommended protocol prior to freezing and storage. All samples were snap frozen in liquid nitrogen and stored at -80oC. Samples were thawed one time and analyzed in duplicate. Plasma glycerol was measured by colorimetric assay kits according to the manufacturer’s recommended protocol (Cayman Chemical, Ann Arbor, MI). Serum lactate brought to room temperature, deprotonated and then measured by fluorescence signals generated by L-Lactate, (Cayman Chemical, Ann Arbor, MI).

Statistics

Dependent t-tests were used to examine differences between ketone-caffeine supplement and water conditions for physical and cognitive performance results. For ketones and glucose, a

repeated measures analysis of variance with condition (ketone/caffeine vs water) and time (11 time points during the testing session) as within factors were used. Pairwise comparisons post hoc was used to examine comparisons between conditions when significant main or interaction effects were present. The alpha level for significance was set at p < 0.05.

Figure 4: Testing Day Protocol

Chapter 4: Results

GI Distress

The supplement was well tolerated. Self- perceived ratings of GI distress were not significantly difference between condition trials. There was only one instance of GI stress other than a 0, and the participant selected a “1” due to eructation of the product, post consumption. In all cases, subject has completed the exercise trial and all additional testing (cognitive testing, blood draws, and finger sticks) was completed as scheduled.

Ketone and Glucose Levels

Capillary blood ketone concentrations at baseline (pre-ingestion) were similar, but all post-supplement consumption values (value #3 and on) were significantly higher than the water condition (p< 0.05; Figure 5). Peak ketones levels typically occurred 15 minutes post ingestion and were more than two-fold higher after supplement ingestion.

Glucose concentrations peaked immediately post-exercise, while gradually returning to baseline levels during the monitored recovery time, however, there were no significant differences between trial conditions (Figure 6).

Time to Exhaustion

Total time to exhaustion was significantly increased by 104 seconds (9.8%) after consumption of the ketone/caffeine supplement (19:33 ±6:57 vs. 17:48 ±6:45 min:sec; t(11)=3.6, p=0.004, d= .02). Eleven of twelve participants increased TTE and the one who showed decreased performance was less than 5 % difference between trials (Table 3). There were no significant correlations between ketones and TTE.

Peak V02

Peak VO2 levels achieved during the supplement consumption was statistically higher than the water control (p < .05). (Table 3)

Wingate Power

Peak power achieved during the Wingate was not statistically different between the ketone/caffeine supplement and water trial (566 ± 114 W and 578 ± 170 W, respectively).

(Table 4). Average power over 30 seconds was not significantly different between the two condition trials (397 ± 24.3 and 394 ± 39.7 W respectively).

Cognitive Testing

There was no significant difference in test scores between conditions.

Rate of Perceived Exertion

There was no significant difference between RPE scores for any of the stages following the ketone/caffeine supplement condition. Each RPE was taken at the end of the five-minute stage.

Respiratory Exchange Ratio

There was no significant difference between RER between conditions. RER increased as the participant continued exercising (Table).

Lactate

There was no statistical difference in plasma lactate between the two conditions, except at the

Pre- Exercise blood measurement, 15 minutes post supplement ingestion (p< .05)

Glycerol

There was no statistical difference between plasma glycerol levels in both ketone/caffeine supplement condition and water control conditions.

Chapter 5: Discussion

Few studies have examined exogenous ketone supplementation on participants of a mixed diet, let alone a keto-adapted cohort. This study extends the findings of our previous study

(Short et al) that showed an ergogenic effect of the same ketone-caffeine supplement in a keto- naïve group by showing a similar performance benefit in a keto-adapted population. This study found significant increase in Time to Exhaustion and an elevated Peak VO2 in the keto-adapted with ketone/caffeine supplement condition over the keto-adapted with water trial. The supplement was well tolerated and did not cause any performance hindering effects. These results support the ergogenic potential of a combined ketone/caffeine supplement for enhancing exercise performance in both keto-adapted and keto-naïve recreationally-trained adult.

The athletes considered for this study were keto-adapted and were selected through an interview process so long as they had been on a chronic ketogenic diet for a time over a several week adaptation period (Volek et al., 2014). This adaptation period will result in the increase of plasma level ketone concentrations to >0.4 mmol/L- 5mmol/L (G. Cahill, 2006; L. Veech,

Britton Chance, Yoshihiro et al., 2001). This can be a considerably trying for individuals to monitor their food adequately enough to transition into nutritional ketosis. The rigor it takes to remain in this state with all the available food options takes much integrity out of an individual let alone an athlete. To ensure that nutritional ketosis was not just a consequence of the 8 hour fasting required with this study, participants were requested to complete a 24 hour extensive food

log stating time of food ate, and their portions. These logs were analyzed by staff research dietician familiar with the well-formulated ketogenic diet. Along with the detailed food logs, and ketone reading, prior to exercise testing with resistance, participants Respiratory Exchange Ratio

(RER) was monitored at onset of warm-up exercise to validate the primary energy substrate utilization for each participant. RER indirectly shows the muscle’s oxidative capacity to get energy from certain substrates through technique of indirect calorimetry (Goedecke et al., 2000).

Participants we assessed during the 4 minute warm-up to see the RER set at onset of exercise. A fat oxidative state, and ideally a keto-adapted individual, will score below .85. The participants were assessed for a final validation of ketosis via this protocol at their baseline testing (RER= .71

±.02). This is the first study of its kind to assess an exogenous ketone/caffeine supplementation on a completely keto-adapted cohort with such pretentious standards. These athletes in themselves are a powerful cohort of self-motivated, unique population.

The ketone/caffeine supplement was able to successfully elevate plasma ketone concentrations. In a keto-naïve population, this ketone/caffeine product was able to significantly increase plasma ketone concentration from baseline (p<.05). Ketone elevation was higher than reported in previous studies due to the already keto-adapted phenotypes of the participants

(Clarke, 2013; Cox et al., 2016). All subjects were provided a standard dose of the ketone/caffeine supplement containing 7.2g of BOHB. Our previous study, currently in review, led to a question at whether a higher dose may result in greater exercise improvements. This answer remains unclear, but the response to higher endogenous level ketones with exogenous

supplementation shows that there is possible cause to review further performance benefits to the already keto-adapted. One recent study reported higher doses up to ~25g BOHB given to participants to increase ketosis showing no significant changes, however, upon further analysis of the product, we found that specific commercially available ketone/caffeine supplement, was reported inappropriately and the BOHB included in the supplement was only 11.7g according to the manufacturer, KetoForce (O’Malley et al., 2017). Thus, leaving the question of higher BOHB administration unanswered. The standard dose of BOHB given to subjects was not analyzed based on d- and l- isoforms of BOHB. Seeing as utilization of each is unclear, these difference may also have a role in the uptake and utilization of exogenous ketones.

In both conditions of the keto-adapted cohort glucose was found to be typically under 90 mg/dL at baseline with gradual decline through the TTE exercise protocol, and then peaked significantly above baseline immediately post anaerobic Wingate testing. Glucose availability declines with increase of stress and elongation of an activity. This pattern is a natural response as glucose typically declines during submaximal exercise, and will then increase during supramaximal exercise due to the hormonal responses that drives hepatic gluconeogenesis

(Wasserman, 1995). We found in our previous study that acute ketosis did not alter glycemic response, which proves a net increase in availability of circulation substrate available for continued or strenuous exercise. As the same effect was seen in a keto-adapted group, we can concur that ketosis will not inhibit hepatic glucose output and result in decrease levels of glucose

that has been reported in previous studies(Mikkelsen, Seifert, Secher, Grøndal, & Van Hall,

2015).

Cycling performance was improved after ingestion of the ketone/caffeine supplement which was proven by the increased ability to cycle longer at incremented resistance of %90 VO2

Peak at baseline and the ability to achieve higher levels of peak VO2. These performance increases parallel with our previous study in review and after a 30 min times trial performance test conducted after an hour of steady state workload at 75%Wmax on a stationary bike (Cox et al., 2016). Results were obtained on a LODE cycle ergometer in a laboratory setting however, the ergogenic effect will translate appropriately to performance environments that are applicable for elongated endurance and sustainable energy due to the efficacy of ketones ability to produce

ATP per unit of oxygen (L. Veech, Britton Chance, Yoshihiro et al., 2001; Sato et al., 1995; The

Western Mirror et al., 2017). During the state of nutritional Ketosis, both acute or chronic, energy can be created quickly and efficiently which would initiate interest in anaerobic performance in a ketogenic state especially in high stress situations where it is found that glucose availability will decrease as the task, both mental and physical, becomes higher in demand

(Mcnay et al., 1999; Yuen et al., 2012). Increased production of ATP will allow for performance to improve with the similar demands of oxygen intake. This is evidenced by the significant increase in peak VO2 found in this study. The ketone/caffeine supplement may prove true for applied positive exercise performance. The endurance aspect of the TTE trial would mimic real

world longevity in sport allowing for the possibility that with an exogenous ketone/caffeine supplement athletes may be able to push athletic ability farther, harder and longer.

The ketone supplement may have had a buffering effect on the lactate associated with high intensity exercise. Although muscle lactate was not measure in this study, we can report that through pairwise comparisons repeated measure we found that within the ketone/caffeine supplement group blood lactate trended higher than the control. This buffering mechanism was suggested in previous studies due to the ability of ketone ester to decrease lactate accumulation in muscle (Cox et al., 2016). This may prove that when the body is in ketosis, ketone utilization by muscle does not appear to be hindered while maintaining glycogen storages. No significant differences were found in serum glycerol, however peaked in both conditions immediately port- exercise indicated accelerated adipose tissue lipolysis, however, seeing as the groups were already metabolically fat-adapted, there would not be much difference with increased plasma ketone levels.

Cognitive test scores showed no significant effects in either control. It was hypothesized that the combine effects of ketones and caffeine would increase cognitive performance after exercise. Ketones have the ability to cross the blood brain barrier and utilized for energy. The idea that when glucose is scarce and competition for energy is high in the skeletal muscle, the brain can utilize ketones allowing for the limited supply of glucose to be used in skeletal muscle for mitigating fatigue. Due to more available energy substrates, improved cognitive function was hypothesized because of the fuel to both brain and body. However, in a ketogenic state, the

brain may be already adapted to ketones as fuel the uptake may be as usual which conclusively would show no changes. Historically, caffeine has been believed to help with basic cognitive functioning based on studies showing improved attention or memory(McLellan, Caldwell, &

Lieberman, 2016), but the standard dose administered may have been too low to see any of the affects.

This caffeine and ketone supplement may have synergistically triggered some benefits as a combined formula. Caffeine research has shown significant impact on exercise performance.

Most doses are higher than the standard dose contained within the supplement (McLellan et al.,

2016). Elongated, graded exercise protocols may not mediate the same effect from caffeine popularly accepted. However, research shows the anaerobic all-out testing portion of our protocol may be the most applicable to caffeine benefits, even in situations such as this where glucose/glycogen storages are depleted (Silva-Cavalcante et al., 2013). Improved TTE may also be due to caffeine ability to decrease RPE (Rodrigues et al., 1990). However, the RPE between the two groups was insignificant, this may have been a playing factor in the 9.8% increase of time. Caffeine has also been reported to increase lipolysis (Ryu et al., 2001) for energy and therefore conserving glucose/glycogen. Caffeine variability is dependent upon dosage, subject, and the testing being done.

No adverse effects were observed from oral administration of the ketone/caffeine supplement from any of the participants. The supplement was well tolerated and safe as reported in other exogenous ketone studies(Clarke, 2013). Self- perceived ratings of GI distress were not

significantly difference between condition trials. There was only one instance of GI stress other than a 0, and the participant selected a “1” due to eructation of the product, post consumption. In all cases, subject has completed the exercise trial and all additional testing (cognitive testing, blood draws, and finger sticks) was completed as scheduled. Recent studies have paired their ketone supplements with 3 liters of supplement, full meals, diet soda, and carbohydrate gels and find “severe participant experiences” but it is undecided if it is due specifically to the exogenous ketone salt supplementation to the volume of consumption prior to the onset of exercise (Clarke,

2013; Leckey et al., 2017). Compared to the 16 ounces provided in this study, we found no indicator of severe distress with the supplement.

Conclusion

In conclusion, ingestion of a ketone/caffeine supplement by a keto-adapted population was well tolerated, successfully elevated ketone concentrations, improved TTE time and increased peak VO2, but had no impact on cognitive function. The supplement used a combination of ketone salts and caffeine, which makes determining the reason for the ergogenic effect difficult. This study was a follow-up from a previous study with a keto-naïve arm that found comparable findings in that cohort as with the keto-adapted. We can conclude that this ketone/caffeine supplement can improve athletic performance but should be studied further to determine optimum dosage for keto-adapted athletes to understand if there is a ceiling effect on the benefits of increased nutritional ketosis.

Bibliography

Balasse, E. O., & Neef, M. A. (1978). Changes induced by exercise in rates of turnover and

oxidation of ketone bodies in fasting man. Journal of Applied Physiology: Respiratory,

Environmental and Exercise Physiology, (44), 5–11.

Brownlow, M. L., Jung, S. H., Moore, R. J., Bechmann, N., & Jankord, R. (2017). Nutritional

Ketosis Affects Metabolism and Behavior in Sprague-Dawley Rats in Both Control and

Chronic Stress Environments. Frontiers in Molecular Neuroscience, 10(May), 1–17.

https://doi.org/10.3389/fnmol.2017.00129

Cahill, G. (2006). Fuel Metabolism in Starvation. Annual Review of Nutrition, 26(1), 1–22.

Cahill, G. F. J., & Veech, R. L. (2003). Ketoacids? Good medicine? Transactions of the

American Clinical and Climatological Association, 114(4), 143–149.

https://doi.org/10.1177/1941738113516075

Carter, M. E., & Brunet, A. (2007). FOXO transcription factors. Current Biology : CB, 17(4),

R113-4. https://doi.org/10.1016/j.cub.2007.01.008

Clarke, K. (2013). NIH Public Accesskinetics, safety and tolerability of (R)-3-hydroxybutyl (R)-

3-hydroxybutyrate in healthy adult subjects, 63(3). 35

https://doi.org/10.1016/j.yrtph.2012.04.008.Kinetics

Cox, P. J., Kirk, T., Ashmore, T., Willerton, K., Evans, R., Smith, A., … Clarke, K. (2016).

Nutritional Ketosis Alters Fuel Preference and Thereby Endurance Performance in Athletes.

Cell Metabolism, 24(2), 256–268. https://doi.org/10.1016/j.cmet.2016.07.010

Dye, J. A., & Chidsey, J. L. (1939). Ketone body-total carbohydrate utilization ratios and their

relation to the problem of ketosis. American Journal of Physiology, (127), 745–750.

Fery, F., & Balasse, E. O. (1985). Ketone body production and disposal in diabetic ketosis: A

comparison with fasting ketosis. Diabetes, 34(4), 326–332.

https://doi.org/10.2337/diab.34.4.326

Féry, F., & Balasse, E. O. (1988). Effect of Exercise on the Disposal of Infused Ketone-Bodies

in Humans. Journal of Clinical Endocrinology & Metabolism, 67(2), 245–250.

Goedecke, J. H., St Clair Gibson, a, Grobler, L., Collins, M., Noakes, T. D., & Lambert, E. V.

(2000). Determinants of the variability in respiratory exchange ratio at rest and during

exercise in trained athletes. American Journal of Physiology. Endocrinology and

Metabolism, 279(6), E1325–E1334. https://doi.org/10.1152/ajpendo.2000.279.6.E1325

Harney, J. P., Gudsnuk, K., Patel, A., Vellipuram, A., Bandaru, S., & Butler, D. (2017).

Endocrine and Reproductive Effects of Ketogenic Diets. (S. Masino, Ed.). Oxford

University Press.

Hashim, S. A., & VanItallie, T. B. (2014). Ketone body therapy: from the ketogenic diet to the 36

oral administration of ketone ester. Journal of Lipid Research, 55(9), 1818–1826.

https://doi.org/10.1194/jlr.R046599

Helge, J. W. (2017). A high carbohydrate diet remains the evidence based choice for elite

athletes to optimise performance. Journal of Physiology, 595(9), 2775.

https://doi.org/10.1113/JP273830

Keller, J., Schmitt, F., Scheff, S., Ding, Q., Chen, Q., Butterfield, D., & Markesbery, W. (2005).

Evidence of increased oxidative damage in subjects with mild cognitive impairment.

Neurology, 64(7), 1152–1156. https://doi.org/10.1212/01.WNL.0000156156.13641.BA

Kennedy, G. (1953). The role of fat depot in the hypothalamic control of food intake in the rate.

Proc R Soc Lond B Biol Sci, (140), 578–596.

Keon, C., Tuschiya, N., Kashiwaya, Y., Sato, K., Clarke, K., Radda, G., & Veech, R. (1995).

Substrate Dependence of the Mitochondrial Energy Status in the Isolated Working Rat

Heart. Biochemical Society TRansactions, 23(2).

L. Veech, Britton Chance, Yoshihiro, R., Chance, B., Kashiwaya, Y., Lardy, H. A., & Cahill, G.

F. (2001). Ketone Bodies, Potential Therapeutic Uses. IUBMB Life (International Union of

Biochemistry and Molecular Biology: Life), 51(4), 241–247.

https://doi.org/10.1080/152165401753311780

Laffel, L. (1999). Ketone bodies: a review of physiology, pathophysiology and application of

monitoring to diabetes. Diabetes/metabolism Research and Reviews, 15(6), 412–426.

https://doi.org/10.1002/(SICI)1520-7560(199911/12)15:6<412::AID-DMRR72>3.0.CO;2-8

Lamanna, J. C., Salem, N., Puchowicz, M., Erokwu, B., Flask, C., & Lee, Z. (2009). Oxygen

Transport to Tissue XXX, 645, 301–306. https://doi.org/10.1007/978-0-387-85998-9

Leckey, J. J., Ross, M. L., Quod, M., Hawley, J. A., & Burke, L. M. (2017). Ketone diester

ingestion impairs time-trial performance in professional cyclists. Frontiers in Physiology,

8(OCT), 1–10. https://doi.org/10.3389/fphys.2017.00806

Maalouf, M., Sullivan, P., Davis, L., Kim, D., & Rho, J. (2007). KETONES INHIBIT

MITOCHONDRIAL PRODUCTION OF REACTIVE OXYGEN SPECIES

PRODUCTION FOLLOWING GLUTAMATE EXCITOTOXICITY BY INCREASING

NADH OXIDATION. Neuroscience, 256–264.

Manninen, A. H. (2004). Metabolic effects of the very-low-carbohydrate diets: misunderstood

"villains" of human metabolism. Journal of the International Society of Sports

Nutrition, 1(2), 7–11. https://doi.org/10.1186/1550-2783-1-2-7

McLellan, T. M., Caldwell, J. A., & Lieberman, H. R. (2016). A review of caffeine’s effects on

cognitive, physical and occupational performance. Neuroscience and Biobehavioral

Reviews, 71, 294–312. https://doi.org/10.1016/j.neubiorev.2016.09.001

Mcnay, E. C., Fries, T. M., & Gold, P. E. (1999). Decreases in rat extracellular hippocampal

glucose concentration associated with cognitive demand during a spatial task. Proc. Natl.

Acad. Sci. U. S. A., 97(6), 2881–2885. https://doi.org/10.1073/pnas.050583697

Mikkelsen, K. H., Seifert, T., Secher, N. H., Grøndal, T., & Van Hall, G. (2015). Systemic,

cerebral and skeletal muscle ketone body and energy metabolism during acute hyper-D-β-

hydroxybutyratemia in post-absorptive healthy males. Journal of Clinical Endocrinology

and Metabolism, 100(2), 636–643. https://doi.org/10.1210/jc.2014-2608

Newman, J. C., & Verdin, E. (2014). Ketone bodies as signaling metabolites. Trends in

Endocrinology and Metabolism, 25(1), 42–52. https://doi.org/10.1016/j.tem.2013.09.002

O’Malley, T., Myette-Cote, E., Durrer, C., & Little, J. P. (2017). Nutritional ketone salts increase

fat oxidation but impair high-intensity exercise performance in healthy adult males. Applied

Physiology, Nutrition, and Metabolism, 42(10), 1031–1035. https://doi.org/10.1139/apnm-

2016-0641

Paoli, A., Bosco, G., Camporesi, E. M., & Mangar, D. (2015). Ketosis, ketogenic diet and food

intake control: A complex relationship. Frontiers in Psychology, 6(FEB), 1–9.

https://doi.org/10.3389/fpsyg.2015.00027

Paoli, A., Grimaldi, K., D’Agostino, D., Cenci, L., Moro, T., Bianco, A., & Palma, A. (2012).

Ketogenic diet does not affect strength performance in elite artistic gymnasts. Journal of the

International Society of Sports Nutrition, 9, 1–9. https://doi.org/10.1186/1550-2783-9-34

Phinney, S. D., Bistrian, B. R., Evans, W. J., Gervino, E., & Blackburn, G. L. (1983). The human

metabolic response to chronic ketosis without caloric restriction: Preservation of

submaximal exercise capability with reduced carbohydrate oxidation. Metabolism, 32(8),

769–776. https://doi.org/10.1016/0026-0495(83)90106-3

Rexford S.A., and D. A. . (2009). NIH Public Access. National Institutes of Health, 37(4), 811–

823. https://doi.org/10.1016/j.ecl.2008.08.005.Brain

Rodger, S., Plews, D., Laursen, P., & Driller, M. (2017). Oral β-hydroxybutyrate salt fails to

improve 4- minute cycling performance following submaximal exercise. J Sci Cycling, 6(1),

26–31. Retrieved from

http://researchcommons.waikato.ac.nz/bitstream/handle/10289/11199/document

%2828%29.pdf?sequence=2&isAllowed=y

Rodrigues, L., Russo, A., Silva, A., Picarro, I., Silva, F., Zogaib, P., & Soares, D. (1990). Effects

of caffeine on the rate of perceived exertion. Braz J Med Bio Research, 23(10), 965–968.

Ruderman, B. N. B., Ross, P. S., Berger, M., & Goodman, M. N. (1974). Regulation of Glucose

and Ketone-Body Metabolism in Brain of Anaesthetized Rats. Biochemical Journal, 138(1),

1–10. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/4275704

Ryu, S., Choi, S., Joung, S., Suh, H., Cha, Y., Lee, S., & LIM, K. (2001). Caffeine as a lipolytic

food component increases endurance performance in rats and athletes. J Nutrion SCience

Vitaminol (Tokyo), 47(2), 139–146.

Sato, K., Kashiwaya, Y., Keon, C., Naotaka, T., King, T., Radda, G., … Veech, R. (1995).

Sato_K_FASEBJ_1995_9_651-8_wo_cover1.pdf.

Shimazu, T., Hirschey, M. D., Newman, J., He, W., Moan, N. Le, Grueter, C. a, … Verdin, E. 40

(2013). NIH Public Access, 339(6116), 211–214.

https://doi.org/10.1126/science.1227166.Suppression

Shivva, V., Cox, P. J., Clarke, K., Veech, R. L., Tucker, I. G., & Duffull, S. B. (2016). The

Population Pharmacokinetics of d-β-hydroxybutyrate Following Administration of (R)-3-

Hydroxybutyl (R)-3-Hydroxybutyrate. The AAPS Journal, 18(3), 678–688.

https://doi.org/10.1208/s12248-016-9879-0

Silva-Cavalcante, M. D., Correia-Oliveira, C. R., Santos, R. A., Lopes-Silva, J. P., Lima, H. M.,

Bertuzzi, R., … Lima-Silva, A. E. (2013). Caffeine Increases Anaerobic Work and Restores

Cycling Performance following a Protocol Designed to Lower Endogenous Carbohydrate

Availability. PLoS ONE, 8(8), e72025. https://doi.org/10.1371/journal.pone.0072025

Stafford, P., Abdelwahab, M. G., Kim, D. Y., Preul, M. C., Rho, J. M., & Scheck, A. C. (2010).

The ketogenic diet reverses gene expression patterns and reduces reactive oxygen species

levels when used as an adjuvant therapy for glioma. Nutrition and Metabolism, 7, 1–11.

https://doi.org/10.1186/1743-7075-7-74

Stubbs, B. J., Cox, P. J., Evans, R. D., Santer, P., Miller, J. J., Faull, O. K., … Clarke, K. (2017).

On the metabolism of exogenous ketones in humans. Frontiers in Physiology, 8(OCT), 1–

13. https://doi.org/10.3389/fphys.2017.00848

The Western Mirror, Cimpian, A., Building, P., Antoun, M., Edwards, K. M., Sweeting, J., …

Strick, P. L. (2017). Serum levels of fatty acid binding protein 4 and fat metabolic markers

in relation to catecholamines following exercise. Clinical Biochemistry, 50(4), 896–902.

https://doi.org/10.1016/j.clinbiochem.2017.05.021

Veech, R. L. (2004). The therapeutic implications of ketone bodies: The effects of ketone bodies

in pathological conditions: Ketosis, ketogenic diet, redox states, insulin resistance, and

mitochondrial metabolism. Prostaglandins Leukotrienes and Essential Fatty Acids, 70(3),

309–319. https://doi.org/10.1016/j.plefa.2003.09.007

Volek, J. S., Freidenreich, D. J., Saenz, C., Kunces, L. J., Creighton, B. C., Bartley, J. M., …

Phinney, S. D. (2016). Metabolic characteristics of keto-adapted ultra-endurance runners.

Metabolism: Clinical and Experimental, 65(3), 100–110.

https://doi.org/10.1016/j.metabol.2015.10.028

Volek, J. S., Noakes, T., & Phinney, S. D. (2014). Rethinking Fat as a fuel for endurance

exercise. European Journal of Sport Science, (15:1), 13–20.

https://doi.org/10.1080/17461391.2014.959564

Wang, K., McCarter, R., Wright, J., Beverly, J., & Ramirez-Mitchell, R. (1991). Regulation of

skeletal muscle stiffness and elasticity by titin isoforms: a test of the segmental extension

model of resting tension. Proceedings of the National Academy of Sciences of the United

States of America, 88(16), 7101–7105. https://doi.org/10.1073/pnas.88.16.7101

Wang, X., Wu, X., Liu, Q., Kong, G., Zhou, J., Jiang, J., … Zhu, Q. (2017). Ketogenic

metabolism inhibits histone deacetylase (HDAC) and reduces oxidative stress after spinal

cord injury in rats. Neuroscience, 366, 36–43.

https://doi.org/10.1016/j.neuroscience.2017.09.056

Wasserman, D. H. (1995). Regulation of glucose fluxes during exercise in the postabsorptive

state. Annual Review of Physiology, 57(32), 191–218.

Wick, N., & Drury, D. R. (1941). P-Hydkoxybutyric. Journal of Biological Chemistry, (138),

129–134.

Yu, Y. M., Tompkins, R. G., Ryan, C. M., & Young, V. R. (1999). The metabolic basis of the

increase of the increase in energy expenditure in severely burned patients. JPEN. Journal

Parenteral Enteral Nutr, 23(3), 160–168. Retrieved from

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&DbFrom=pubmed&Cmd=Link&Li

nkName=pubmed_pubmed&LinkReadableName=Related

Articles&IdsFromResult=10338224&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.

Pubmed_ResultsPanel.Pubmed_RVDocSum%5Cnhttp://www.ncb

Yuen, E. Y., Wei, J., Liu, W., Zhong, P., Li, X., & Yan, Z. (2012). Repeated Stress Causes

Cognitive Impairment by Suppressing Glutamate Receptor Expression and Function in

Prefrontal Cortex. Nueron., 73(5), 962–977.

https://doi.org/10.1016/j.neuron.2011.12.033.Repeated

Zajac, A., Poprzecki, S., Maszczyk, A., Czuba, M., Michalczyk, M., & Zydek, G. (2014). The

effects of a ketogenic diet on exercise metabolism and physical performance in off-road

cyclists. Nutrients, 6(7), 2493–2508. https://doi.org/10.3390/nu6072493

Appendix A: Figures

Figure 5: Capillary blood beta-hydroxybutyrate responses

Ketone levels were assessed via blood from individual finger sticks at 11 different time points. (*) Indicates significantly different (p<0.05) from corresponding time point for water. (#) Indicates significantly different (p<0.05) than baseline value for respective condition.

Figure 6: Capillary blood glucose responses

Glucose levels were assessed via blood from individual finger sticks at 11 different time points. No statistically significant difference was observed at any point between conditions. (#) Indicates significantly different (p<0.05) than baseline value for both conditions

Figure 7: Individual responses in time to exhaustion trial

The time cycled post-ketone/caffeine supplementation was significantly higher (p<0.01) determined via two-tailed t-test.

Appendix B: Tables

VO2max Caffeinated Sex Age Weight (kg) Height (cm) (ml/kg/min) Beverages / wk Female 30 71 165 31.3 21 Male 32 86.4 181.3 39 0 Male 42 88 173.9 40.5 14 Male 27 91.25 180 39.8 14 Male 40 77.3 184 64.3 14 Female 31 75.2 159.5 21.8 28 Male 36 83.4 176.9 38.8 14 Female 45 92.3 182.7 27.2 35 Male 49 84.4 185.9 40.3 7 Male 33 84.5 175.9 39.1 21 Male 26 80.4 178.7 45.3 35 Male 42 71.6 178.7 44.2 0 Mean ± SD 36.08 ± 7.5 83.6 ± 9.67 176.9 ± 7.68 40.03 ± 10.47 16.9 ± 11.7

Table 2: Participant Characteristics One caffeinated beverage was equal to 8oz.

Ketone Supplement Water Peak Ketone Peak VO2 Peak VO2 % TTE % VO2 Participant (mM) TTE (min:sec) (ml/kg/min) TTE (min:sec) (ml/kg/min) Change Change 1 2.6 15:24 30.2 10:10 30.2 51.5% 0% 2 1.6 21:06 43..3 18:46 23.7 12.5% 82.7% 3 1 21:07 43.7 28:58 23.5 11.1% 85.9% 4 1.6 17:17 42.3 21:39 41.8 2.2% 1.19% 5 2.3 23:18 66.9 15:00 63.7 5.0% 5.0% 6 1.2 22:40 26.4 12:15 26.5 26.9% -.3% 7 1.6 23:34 43.6 16:09 39.3 -3.8% 10.9% 8 1.5 26:24 25.9 10:22 23.5 17% 10.2% 9 1.1 22:46 48 30:15 45 8% 6.6% 10 2.8 15:56 37.6 18:48 36.9 4.9% 1.8% 11 0.7 24:09 47.6 11:13 42.7 .4% 11.4% 12 1.7 21:01 45.2 20:00 44.8 4.8% 1.5% 18.1 ± Mean 1.64 ± 0.64 19:19 ± 6:57* 41.75 ± 3.2* 17:48 ± 6:44* 36:8 ± 3.5 11.7 ± 15 31.2*

Table 3: Peak beta-hydroxybutyrate levels and TTE and VO2 responses Data (mean + standard error; n = 12) for TTE was statistically different (*) (p<0.01) determined via two-tailed t-test. A trend for higher VO2 after ketone/caffeine supplement consumption (p=0.08) was determined via two-tailed t-test.

Ketone Supplement Water % Peak Peak Average Peak Average Power % Average Participants Power (W) Power (W) Power (W) Power (W) Change Power Change 1 422 279 338 249 24.8% 12% 2 694 518 734 523 -5.4% -0.9% 3 563 449 734 521 -23.2% -13.8% 4 589 396 560 351 5.1% 12.9% 5 728 385 567 429 28.4% -10.1% 6 308 209 226 174 36.4% 20.1% 7 536 418 511 406 4.9% 3% 8 518 378 490 160 5.7% 135.9% 9 613 490 605 461 1.2% 6.2% 10 564 418 782 592 -27.8% -29.4% 11 629 393 749 450 16% -12.8% 12 622 436 634 413 -1.9% 5.7% Mean 566 ± 114 397 ± 24.3 578 ± 170 394 ± 39.7 2.68 ± 19 10.8 ± 41.8

Table 4: Wingate power output responses Wingate testing occurred immediately after TTE testing and data (mean + standard error) showed no significant difference in average power within the conditions and no significant difference found in overall peak power.

Table 5: Respiratory exchange ratio responses