Health and Social Development, Faculty of (Okanagan)
Health and Exercise Sciences, School of (Okanagan)
DSpace
UBCO
Tortoriello Romero, Juan Pablo
Master of Science - MSc
University of British Columbia
The potential therapeutic effects of ketosis have recently garnered attention in the scientific community and lay public. Although typically achieved by fasting or following a restrictive very-low carbohydrate diet, ketosis can now be achieved through exogenous ketone supplements, which have been shown to lower glucose and improve heart function, making them a potential therapeutic option in type 2 diabetes. However, studies conducted to date are short-term and many questions remain related to feasibility of longer-term exogenous ketone supplementation. The purpose of this research was to conduct a remote randomized double-blind, placebo-controlled pilot trial to assess the feasibility of 90 days of exogenous ketone supplementation in people living with type 2 diabetes. Twenty individuals living with type 2 diabetes were randomized to consume an exogenous ketone supplement (n=10, 59 mL of flavored concentrate containing 5 g free beta-hydroxybutyric acid + 5 g of the ketone precursor 1,3-butanediol) or a volume, taste- and pH-matched placebo (n=10) three times per day for 90 days. All trial procedures were conducted remotely. The main feasibility outcomes were recruitment rate, compliance/adherence, and participant retention/completion of the trial. Preliminary efficacy outcomes included blood glucose regulation (hemoglobin A1c, continuous glucose monitoring), cardiovascular disease risk factors (waist circumference, blood pressure, blood lipids), and general health questionnaires (physical activity, general health perception, food cravings, and dietary intake). Twenty participants were recruited primarily through specialized third-party social media advertising across ~6 months. Four participants dropped out of the exogenous ketone group due to gastrointestinal issues (n=2) or urge to vomit (n=2) and one participant dropped out of the placebo group due to drink taste. The remaining participants self-reported adherence to thrice daily drink consumption which was generally high at >70% across all weeks of the study. Remote data collection was largely feasible. Acceptability and general health questionnaires showed no obvious differences between groups. Overall, 90 days of exogenous ketone supplementation showed mixed feasibility. High drop-out due to gastrointestinal issues in the ketone group suggests that an alternative supplement formulation would likely be needed for future long-term adherence. However, participants who remained in the trial did show good compliance.
EXAMINING THE FEASIBILITY OF PROLONGED KETONE SUPPLEMENT DRINK CONSUMPTION IN ADULTS LIVING WITH TYPE 2 DIABETES by Juan Pablo Tortoriello Romero M.D. Universidad Anáhuac Mexico | Norte, 2019 A THESIS SUBMITED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE COLLEGE OF GRADUATE STUDIES (Health and Exercise Sciences) THE UNIVERSITY OF BRITISH COLUMBIA (Okanagan) December 2024 © Juan Pablo Tortoriello Romero, 2024 ii The following individuals certify that they have read, and recommend to the College of Graduate Studies for acceptance, a thesis entitled: Examining the Feasibility of Prolonged Ketone Supplement Drink Consumption in Adults Living with Type 2 Diabetes submitted by Juan Pablo Tortoriello Romero in partial fulfillment of the requirements of the degree of Master of Science. Dr. Jonathan P. Little, School of Health and Exercise Sciences_________________________________ Supervisor Dr. Sarah Purcell, School of Health and Exercise Sciences____________________________________ Supervisor Committee Member Dr. Darren Candow, University of Regina________________________________________________ Supervisor Committee Member Dr. Brodie Sakakibara, University of British Columbia (Vancouver)____________________________ University Examiner iii Abstract The potential therapeutic effects of ketosis have recently garnered attention in the scientific community and lay public. Although typically achieved by fasting or following a restrictive very-low carbohydrate diet, ketosis can now be achieved through exogenous ketone supplements, which have been shown to lower glucose and improve heart function, making them a potential therapeutic option in type 2 diabetes. However, studies conducted to date are short-term and many questions remain related to feasibility of longer-term exogenous ketone supplementation. The purpose of this research was to conduct a remote randomized double-blind, placebo-controlled pilot trial to assess the feasibility of 90 days of exogenous ketone supplementation in people living with type 2 diabetes. Twenty individuals living with type 2 diabetes were randomized to consume an exogenous ketone supplement (n=10, 59 mL of flavored concentrate containing 5 g free beta-hydroxybutyric acid + 5 g of the ketone precursor 1,3-butanediol) or a volume, taste- and pH-matched placebo (n=10) three times per day for 90 days. All trial procedures were conducted remotely. The main feasibility outcomes were recruitment rate, compliance/adherence, and participant retention/completion of the trial. Preliminary efficacy outcomes included blood glucose regulation (hemoglobin A1c, continuous glucose monitoring), cardiovascular disease risk factors (waist circumference, blood pressure, blood lipids), and general health questionnaires (physical activity, general health perception, food cravings, and dietary intake). Twenty participants were recruited primarily through specialized third-party social media advertising across ~6 months. Four participants dropped out of the exogenous ketone group due to gastrointestinal issues (n=2) or urge to vomit (n=2) and one participant dropped out of the placebo group due to drink taste. The remaining participants self-reported adherence to thrice daily drink consumption which was generally high at >70% across all weeks of the study. Remote data collection was largely feasible. Acceptability and general health questionnaires showed no obvious differences between groups. Overall, 90 days of exogenous iv ketone supplementation showed mixed feasibility. High drop-out due to gastrointestinal issues in the ketone group suggests that an alternative supplement formulation would likely be needed for future long-term adherence. However, participants who remained in the trial did show good compliance. v Lay summary Ketones are naturally produced by our bodies during starvation or when following a very low-carbohydrate diet. Scientists have recently created exogenous ketones, which allow humans to raise blood ketones by consuming a drink. Initial studies show that drinking ketones can lower blood sugar, suggesting that ketone drinks could potentially aid in the treatment of type 2 diabetes. However, ketone drinks have a notoriously bad taste, so it is questionable if people living with type 2 diabetes can realistically consume them over many weeks/months. This research sought to determine if it was feasible for people living with type 2 diabetes to consume ketone drinks three times per day over 90 days. The results were mixed as 4 out of 10 people dropped out of the ketone drink group due to taste and tolerability issues, yet the remaining 6 people who stayed in the study showed good compliance. vi Preface The following individuals were involved in the design, conduction, execution, analysis, and writing of this thesis. • Dr. Jonathan Little: Principal Investigator leading the Exercise Metabolism and Inflammation Laboratory was primarily responsible for the initial conceptualization of the trial. As the author’s supervisor, Dr. Little’s help with troubleshooting and guidance was always readily available. Dr. Little secured the funding from the Canadian Institutes of Health Research (CIHR) to fund the study, designed the overall concept, provided oversight for data analyses, and edited/revised the thesis. • Dr. Kaja Falkenhain: Postdoctoral Fellow at Pennington Biomedical Research Centre. Dr. Falkenhain was primarily responsible (while she was a PhD student at UBCO) for the development of the detailed trial protocol based off the CIHR grant awarded to Dr. Little. She crafted the initial ethics submission and trial registration and was involved in the early stages of the trial until the author took over as lead researcher. • Dr. Alexis Marcotte-Chenard: Postdoctoral Fellow in the Exercise Metabolism and Inflammation Laboratory. Dr. Marcotte-Chenard guided and supported data analysis for this trial. • Dr. Barbara Oliveira: Research and Administration Manager at the Centre for Chronic Disease Prevention and Management. Dr. Oliveira actively contributed to the design of this study. • Helena Neudorf: PhD Candidate, actively contributed to the design of this study. • MSc Student Victoria Bouck: Conducted initial data cleaning and analysis related to continuous glucose monitoring outcomes. • MSc Student Juan Pablo Tortoriello Romero (author). Partially responsible for the design of the trial, aided with ethics and clinical registrations, executed the study from participant vii recruitment to trial completion and/or participant withdrawal, in charge of logistics for product delivery, participant remuneration, and overall coordination of the trial, collected all data and aided participants in troubleshooting during the trial, led data analyses under supervision of Drs. Little, Marcotte-Chenard, and Falkenhain, and is the main author who wrote the first draft of this thesis, edited/revised by Dr. Little. The present study was approved by the University of British Columbia Clinical Research Ethics Board (UBC CREB number: H22-00644). The study conformed to the standards set by the Declaration of Helsinki, and was pre-registered (NCT05983562). The author declares no use of artificial intelligence in this work. viii Table of contents Abstract ...............................................................................................................................................iii Lay summary ....................................................................................................................................... v Preface ................................................................................................................................................. vi Table of contents ............................................................................................................................... viii List of tables .........................................................................................................................................x List of figures ....................................................................................................................................... xi Introduction and background .............................................................................................................. 1 Type 2 diabetes ............................................................................................................................... 1 Type 2 diabetes pathophysiology .................................................................................................... 1 Current treatment paradigms ......................................................................................................... 6 Type 2 diabetes and public health .................................................................................................. 8 Ketones: biochemistry, physiology, and research development ..................................................... 8 Exogenous ketosis: the idea, the development, the directions .................................................... 11 Exploring feasibility ....................................................................................................................... 15 Objectives: ......................................................................................................................................... 18 Primary Objective: ......................................................................................................................... 18 Criteria for Determining Feasibility (Primary Outcome): .......................................................... 18 Secondary Objectives: ................................................................................................................... 18 Methods ............................................................................................................................................ 21 Ethical approval: ............................................................................................................................ 21 Sample size: ................................................................................................................................... 21 Inclusion criteria: ........................................................................................................................... 21 Exclusion criteria: .......................................................................................................................... 21 Assessment tools: .......................................................................................................................... 22 Study flow:..................................................................................................................................... 26 Product selection and dose prescription ................................................................................... 26 Supplement details ........................................................................................................................ 27 Shipment and delivery to participants ...................................................................................... 28 Recruitment and randomization ............................................................................................... 28 Study design: ............................................................................................................................. 30 Study timeline overview ............................................................................................................ 30 ix Data management and participant security: ............................................................................. 32 Statistical Analyses .................................................................................................................... 33 Results ............................................................................................................................................... 34 Data presented as mean ± standard deviation. ............................................................................ 37 Supplementation compliance ....................................................................................................... 37 Secondary outcome completion rate ............................................................................................ 38 Preliminary efficacy ....................................................................................................................... 40 Discussion .......................................................................................................................................... 50 Overview ....................................................................................................................................... 50 Recruitment methods, rate, and randomization ........................................................................... 50 Compliance, feasibility, and acceptability ..................................................................................... 52 Assessment tools completion rates ............................................................................................... 53 Preliminary efficacy ....................................................................................................................... 55 Strengths, limitations, and future directions................................................................................. 55 Conclusion ......................................................................................................................................... 57 Bibliography ....................................................................................................................................... 59 Appendices ........................................................................................................................................ 64 Appendix A. Health screening questionnaire ................................................................................ 64 Appendix B. Theory of planned behavior questionnaire .............................................................. 65 Appendix C. Drink acceptability questionnaire ............................................................................. 68 Appendix D. Godin physical activity questionnaire ....................................................................... 69 Appendix E. EQ-5D-5L questionnaire ............................................................................................ 70 Appendix F. Food cravings questionnaire. ..................................................................................... 71 Appendix G. VAS Hunger and Fullness .......................................................................................... 72 Appendix H. Overall acceptability questionnaire .......................................................................... 73 x List of tables Table 1. Overview of exogenous ketone supplements currently available in the market................. 12 Table 2. Sample characteristics and cardiovascular and anthropometric records ............................ 37 Table 3. Participants’ rate of compliance/fulfillment of requested outcomes for the study divided by timepoint ........................................................................................................................................... 40 Table 4. Preliminary effect produced by the consumption of thrice a day for 90 days exogenous ketone supplement compared to a non-caloric taste matched placebo upon blood chemistry levels focused for lipidic profile ................................................................................................................... 42 Table 5. Preliminary effect produced by the consumption of thrice a day for 90 days exogenous ketone supplement compared to a non-caloric taste matched placebo upon blood cell count and inflammatory markers ....................................................................................................................... 43 Table 6. Preliminary effect produced by the consumption of thrice a day for 90 days exogenous ketone supplement compared to a non-caloric taste matched placebo upon anthropometric records as waist circumference ...................................................................................................................... 44 Table 7. Preliminary effect produced by the consumption of thrice a day for 90 days exogenous ketone supplement compared to a non-caloric taste matched placebo cardiovascular indicators as systolic and diastolic blood pressure ................................................................................................. 45 Table 8. Mean scores for theory of planned behavior visual analog scale (VAS) survey related to the timepoint and condition of the intervention ................................................................................... 45 Table 9. Mean scores for Godin 7 day physical activity survey related to the timepoint and condition of the intervention ............................................................................................................................ 45 Table 10. Mean scores for Hunger visual analog scale (VAS) survey related to the timepoint and condition of the intervention ............................................................................................................ 46 Table 11. Mean scores for Fullness visual analog scale (VAS) survey related to the timepoint and condition of the intervention ............................................................................................................ 46 Table 12. Mean scores for food cravings survey related to the timepoint and condition of the intervention ....................................................................................................................................... 46 Table 13. Mean scores for EQ-5D-5L VAS survey related to the timepoint and condition of the intervention ....................................................................................................................................... 47 Table 14. Mean scores for drink acceptability survey related to the timepoint and condition of the intervention ....................................................................................................................................... 47 Table 15. Mean scores for gastrointestinal distress survey related to the timepoint and condition of the intervention ................................................................................................................................ 47 Table 16. Results for ASA24 dietary assessment related to timepoint and condition of the intervention ....................................................................................................................................... 48 Table 17. Preliminary effect produced by the consumption of thrice a day for 90 days exogenous ketone supplement compared to a non-caloric taste matched placebo upon readings for continuous glucose monitor metrics .................................................................................................................... 49 xi List of figures Figure 1. The T2D triumvirate, a simplistic yet accurate first approach to understanding T2D pathophysiology .................................................................................................................................. 3 Figure 2. Schematic representation of the ominous octet, with current pharmacological treatments indicated for each pathophysiological process ................................................................................... 6 Figure 3. Participant recruitment flow chart for randomization ....................................................... 29 Figure 4. Graphical representation of trial flow ................................................................................ 32 Figure 5. Consort flow chart for participant recruitment, screening, enrollment, allocation, and trial completion/withdrawal ..................................................................................................................... 36 Figure 6. Participant compliance to supplement consumption ........................................................ 38 Introduction and background Type 2 diabetes Type 2 diabetes (T2D) is a metabolic condition characterized by chronically high blood glucose levels secondary to the (inter)action of multiple pathophysiological processes that mainly affect the correct functioning and production of the hormone insulin (1). The major physiological role of insulin is to facilitate the transportation of circulating glucose into cells and tissues where energy is needed. Insulin also promotes the storage of glucose in the liver and the muscle tissue in the form of glycogen, and it counteracts the endogenous production of glucose (gluconeogenesis) (2). In patients with T2D, the production of insulin by the beta cells of the pancreas is impaired. In addition, muscle glucose uptake is decreased, and, partly due to the lack of counterregulatory action by the decreased insulin production, there is excess endogenous glucose production by the liver. The combination of these three main factors will develop into a pathological state that leads to a chronic increase in circulating blood glucose (3). This “triumvirate” was initially coined in 1988 by Dr. Ralph DeFronzo, distinguished researcher responsible for many seminal advances in the understanding and treatment of T2D (3). However, himself, complementing his own discoveries and the vast clinical and laboratory research that has been made, a couple of decades later, redefined and reshaped T2D pathophysiology describing up to eight different mechanisms interacting in the complex evolution of this condition, expanding the triumvirate to the “ominous octet” of T2D pathology and opening discussion for the paradigm that still guides treatment today (4). Type 2 diabetes pathophysiology The previous definition of T2D pathophysiology is concise and accurate. In general, this condition can be understood as a vicious cycle, where there is an overproduction of insulin by the 2 beta cells in the pancreas as an attempt to compensate for the prevailing degree of insulin resistance, which leads to hyperinsulinemia and further development of metabolic dysfunction. Insulin resistance in the liver results in a lack of suppression of hepatic glucose production, which will perpetuate elevated blood glucose that drives increased insulin production. This situation is compounded by insulin resistance in the muscle (and other peripheral tissues) leading to less insulin-stimulated glucose uptake and a further exacerbation of hyperglycemia (5). Figure 1 depicts the basic mechanisms that are altered in T2D, which are recognized to present concurrently, contributing to a vicious cycle. Under healthy circumstances, these three physiological processes would counter-regulate each other and create a harmonious and even simple equilibrium between glucose absorption, insulin production, and systemic blood glucose homeostasis. However, as mentioned above, the dysfunction in physiological actions and regulatory interactions creates a disequilibrium characterized by high blood glucose levels, constant stimulation of insulin secretion from the pancreatic beta cells, and reduced insulin action (i.e., insulin resistance) in various tissues. The beta cells appear to be able to compensate for insulin resistance (via increased insulin secretion) for a certain period, until chronic hyperglycemia prevails, leading to a diagnosis of diabetes based on blood glucose concentrations (6). By the time of T2D diagnosis, the constant extra stress to the beta cell is such that the reserve of pancreatic function is limited, beta cell volume and mass are decreased, and this compensatory effect is no longer enough to regulate systemic glucose levels. Strikingly, it has been estimated that beta cell function has decreased up to 50% by the average time a T2D diagnosis is made (7). 3 Figure 1. The T2D triumvirate, a simplistic yet accurate first approach to understanding T2D pathophysiology. During initial states, the lack of insulin action will be counter regulated by an increased yet insufficient production of this hormone. The lack of action/sensitivity to insulin will be evidenced by an absence of counterregulatory mechanisms that should decrease systemic blood glucose levels. Adapted from the ideas of De Fronzo (4) and created with BioRender.com Widening the pathophysiological features of T2D, and further analyzing the effects that the T2D triumvirate has in other systems and tissues, many other organs and organ systems are now implicated in the development and progression of T2D, as depicted in Figure 2 (3) (8). • Obesity, adipocytes, and lipotoxicity: People living with overweight and obesity have an increased volume of dysfunctional enlarged adipocytes that are poor at storing lipid and instead can create a state of lipotoxicity in other tissues. In physiological conditions, insulin controls the degradation of stored lipids (triglycerides) into free fatty acids (FFA) that will enter systemic circulation. There is evidence of resistance to this anti-lipolytic effect by adipose tissue, which will cause a sustained increase in the levels of FFA; this in turn causes a natural increase in gluconeogenesis, thus creating a new vicious cycle in the context of T2D. In addition, the high levels of FFA create a disharmonious functioning of the 4 inflammatory cells decreasing the amount of insulin-sensitizing adipokines and creating a constant state of inflammation. As excess fat builds up in the liver, pancreas, and muscle tissue, it will decrease insulin sensitivity and impair insulin secretion by the pancreatic beta cells. In the muscle, this state of lipotoxicity and consequential insulin resistance contributes to defective insulin signaling to GLUT4, thus reducing muscle glucose uptake. Reducing the size of adipose tissue (adipocytes) via weight loss is believed to counteract this excess lipolysis and FFA deposition in various tissues, thus improving T2D pathophysiology (4) (9). • The gut, endocrinology and incretins: Two main hormones (incretins) produced in the gut have major implications in blood glucose control by stimulating the production/or suppression of the hormones related to it: gastric inhibitory polypeptide (GIP) and glucagon-like peptide-1 (GLP-1). GIP has a direct effect on the pancreatic tissue, stimulating the secretion of insulin, which is impaired in T2D. GLP-1 has similar actions and also decreases glucagon production, which helps to reduce hepatic glucose production. Added to this, incretins have direct effects in the gut such as delayed gastric emptying, which significantly decreases the rise of postprandial blood glucose levels. Medications for T2D such as GLP-1 receptor agonists or DPP-4 antagonists (which block the degradation of incretins) have been proven effective and are a popular line of treatment (4) (10). • Glucagon, the per excellence counterregulatory hormone: Whilst the pancreatic beta cells produce insulin, the pancreatic α-cells produce glucagon, which acts as a direct counterregulatory hormone having basically opposite effects to insulin. There is solid evidence of an increased level of fasting systemic glucagon plus a hypersensitivity to this hormone in people living with T2D (4) (11). 5 • The kidney and glucose reabsorption: Within the most recent approved drugs for T2D treatment are SGLT2 inhibitors. This medication blocks glucose reabsorption by the SGLT2 transporter in the kidney. Patients with T2D have a state of hyperreactive activity of this transporter and thus increasing glucose reabsorption while not eliminating it through urine (4) (12). • The nervous system and its complexity: This system has been studied in the context of T2D and in overweight and obesity. It is suggested that the obesity - and hence the T2D -epidemic may be importantly influenced by the brain and within the proposed mechanisms, a delayed response to hyperinsulinemia-mediated appetite suppression in the hypothalamus. There is a strong connection between gut hormones (incretins), previously mentioned, and these pathways in the central nervous system (4) (13). 6 Figure 2. Schematic representation of the ominous octet, with current pharmacological treatments indicated for each pathophysiological process. Redrawn and adapted from DeFronzo 2009 and Thrasher 2017. Created with BioRender.com Current treatment paradigms As already stated, T2D diabetes is a multifactorial disease which chronic complications are responsible for the main part of the decrease in health and life expectancy in people living with this disease. In mild newly diagnosed cases of T2D of pre-diabetes (a state of hyperglycemia where Hb A1c levels range from 6.0%-6.4%) the first line of treatment will be related to lifestyle interventions: physical activity and nutrition. If these changes in patient’s lifestyle prove inefficient or uncapable of being performed several medications are readily available for physicians (14). • First line of medication treatment: 7 o Metformin: Safe and cost effective, increases insulin sensitivity and decreases hepatic glucose output. Potential side effects include nausea, diarrhea, and “metallic” taste. • Second line of medication treatment: o DPP-4 inhibitors: Increase postprandial insulin production. Medications that increase insulin production can be associated with an increase in long term pancreatic beta cells stress and hence decrease in their functionality. o GLP-1 receptor agonists: Mainly decrease digestion rates and reduce appetite, hence weight loss effect is the main responsible for enhanced glucose control. A common side effect is nausea. o SGLT2 inhibitors: This medication reduces glucose reabsorption in the kidney, eliminating greater rates of glucose through urine. Side effects are usually related to urinary tract infections. o Insulin secretagogues: Increase pancreatic beta cell insulin output. They are associated with higher risk of hypoglycemia and long term reduced pancreatic function. o Thiazolidinediones: Increase insulin sensitivity. Important side effects include weight gain and increased risk of heart failure. o Insulin therapy: Injected insulin will compensate for a lack of endogenous insulin production or a decrease in insulin sensitivity. Its main side effects or complications are the administration method (injected) and higher risks of hypoglycemia. The purpose of this section on current T2D medication treatments is not to discount their efficacy, but to rationalize that even though the options are multiple, each option has positive and 8 potentially negative outcomes with treatment decisions individualized and constantly optimized. Hence, even with the significant advances in medication management of T2D, there is still a good margin for improvement of current lines of treatment and development of new strategies that can enhance glucose control of people living with this disease. Type 2 diabetes and public health In the last decades, T2D has become a public health concern, being referred to as “one the fastest growing global health emergencies of the 21st century” (15) by the International Diabetes Federation and high blood glucose being “the third highest risk factor for premature mortality” (16) according to the World Health Organization. Worldwide, the social, economic and health impact of T2D is therefore not surprising, with a world prevalence estimated at 6.28% and an alarming increase in the incidence of this disease across most nations (17). In Canada, T2D prevalence is being forecasted to increase 44% in the decade comprising 2015-2025, from a net prevalence of 9.3% in 2015, to 12.1% predicted for 2025, according to Diabetes Canada estimates (18). The cost of treating T2D, which typically requires long-term drug treatment and constant monitoring, is also increasing from an estimated $32 billion CAD in direct costs in 2023 to over $39 billion CAD by 2028 (18). Given that lowering blood glucose is the major treatment target for individuals living with T2D, alternative glucose lowering options are of interest, with ketone supplements emerging as a potential therapeutic strategy (19) (20). Ketones: biochemistry, physiology, and research development Ketone bodies are produced endogenously during periods of fasting, low calorie intake and/or when following a very-low carbohydrate diet. During these states, acetyl CoA levels in the liver will increase by means of fatty acid β-oxidation. Two molecules of acetyl CoA will react to form a molecule of acetoacetyl CoA via the enzyme acetoacetyl-CoA thiolase. A third molecule of Acetyl 9 CoA can then react with acetoacetyl CoA to form 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) catalyzed by the enzyme HMG-CoA synthase. This last reaction is considered rate-limiting for endogenous production of ketone bodies and, interestingly, is mediated by a protein called FOXO2A, which is upregulated by glucagon and downregulated by insulin. HMG-CoA in the cytosol will be a precursor to cholesterol and within the mitochondria is degraded to a molecule of Acetyl CoA plus a molecule of acetoacetate; acetoacetate is the first of three molecules commonly referred to as ketone bodies. Acetoacetate will either suffer spontaneous degradation and be eliminated by the respiratory system (breath) in the form of acetone (a second ketone body) or by means of the enzyme β-hydroxybutyrate dehydrogenase (BDH) be oxidized into β-hydroxybutyrate (β-HB) (21). β-HB is the most abundant circulating ketone body and can be oxidized by vital tissues such as the brain, the heart, the kidneys and the muscle. In the presence of adequate carbohydrate intake, glucose molecules cross the blood brain barrier (BBB) and are believed to be the major metabolic substrate for brain ATP production. However, in a state of ketosis, β-HB can readily cross the BBB and, through the reverse chain of reactions described above, ketone bodies can provide acetyl CoA for ATP production as an alternate fuel to carbohydrate (22) (23). This broad overview of a complex chain of biochemical reactions allows us to understand two things that may explain the growing research and popular interest in ketones: 1) the evolutionary-conserved process of ketosis that can provide the brain fuel during times of starvation or low energy intake; and 2) the potential implications and excitement surrounding the development of exogenous sources of ketone bodies. Research on ketosis dates back to at least the early 20th century, when researchers correlated the positive effects that fasting had in the treatment of epilepsy with the increase of ketonemia that resulted from it (24). Dr. Russell Wilder proposed then that the same effects of long term fasting could be obtained from what became to be known as the ketogenic diet (KD), consisting of a very-low carbohydrate intake and a high fat consumption, “forcing” the body’s metabolism to obtain its 10 caloric uptake from fat oxidation and hence producing ketone bodies for the vital organs such as the brain (25). Science and pharmacological therapies advanced and epilepsy treatments drifted away from this ketogenic nutritional strategy. Recent research and the development of research techniques such as ketone esters infusion in the context of parenteral nutrition (26), have set the tone for the continuous curiosity of the potential effects of ketones in human health and disease. Ketone bodies and KD have been widely studied and associated with (19): • Weight loss: KD promotes fat loss through several mechanisms, including reduced appetite, increased fat oxidation, and enhanced satiety. • Metabolic Syndrome: KD has been shown to improve markers of metabolic syndrome, such as dyslipidemia, hypertension, and abdominal obesity. • Neurodegenerative diseases: emerging evidence suggests that ketone bodies may benefit conditions such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis (ALS) by providing neuroprotective effects and enhancing brain mitochondrial function. • Cancer: ketone bodies have been investigated as an adjunctive treatment for certain types of cancer, based on the hypothesis that cancer cells have impaired mitochondrial function and rely heavily on glucose for energy. By limiting glucose availability and increasing ketone bodies, a KD may alter metabolism of cancer cells while providing energy to healthy cells. Measurable blood ketone body concentration under a normal diet will range within 0.2-0.5 mmol/L (23). Consensus varies when trying to define the desirable levels of ketosis, and which is the best nutritional strategy to achieve it. Over the course of three days, a moderate restriction in dietary carbohydrate (e.g,. <130 g/day carbohydrates) seems to have no impact in the onset of ketosis; a low-carbohydrate diet (often defined as 50-130 g/day carbohydrates) can raise β-HB levels to ~0.3 mmol/l whereas very low-carbohydrate diets (<50g/day carbohydrates) can raise β-HB levels to 11 levels above 1.0 mmol/l (27). These concentrations will vary within individuals and measuring techniques/tools, however it is useful to understand that small increases in circulating ketone concentrations are already considered a ketogenic state. Exogenous ketosis: the idea, the development, the directions The history of exogenous ketones development, beyond an intravenous infusion medical treatment, is interesting. In 2003, the Defense Advanced Research Projects Agency (DARPA), launched a request for developing “a novel, energetically efficient food that would allow soldiers to maintain their physical and mental performance over five days on a battlefield” (28). In response to this, Professor Kieran Clarke from the University of Oxford and Dr. Richard Veech†, with an initial $10 million USD grant, teamed up to develop a meal supplement drink based on the ketone monoester ((R)-3-hydroxybutyl (R)-3-hydroxybutyrate that had been infused intravenously in research and medical practice. After years of intense research and development, safety and tolerability of this supplement was demonstrated in a human study (23). The ketone monoester drink developed was capable of increasing peak levels of circulating β-HB to >3 mmol/l within 1-2 hours at a dose of 714 mg/kg, which was well tolerated with no identified safety issues (23). This research set the basis for what is today a successful international company founded by Prof. Clarke (28) and a separate commercial entity started by Dr. Veech (29) both of which sell the original ketone monoester primarily marketed as a sports drink to enhance physical performance. Currently, multiple commercial brands of ketone supplements are sold worldwide. Research and industry development has led to many different formulations, which include the original ketone monoester, ketone salts wherein ketones (B-HB) are bonded to a mineral (e.g. Sodium, Potassium, Magnesium), free R-β-hydroxybutyric acid, various ketone precursors (e.g., R-1,3-butanediol and medium-chain triglycerdies), and various combinations thereof. The ketone monoester and ketone precursors are usually sold in liquid presentation ready to drink, while ketone salts are often a powder presentation 12 to be dissolved in water before consumption. An overview of various commercial ketone supplements that are currently available is presented in Table 1. Table 1. Overview of exogenous ketone supplements currently available in the market. General overview Potential to increase blood [ketone] Ketone salts -Racemic mixture of BHB molecules (R,S - BHB) that is attached to a mineral molecule such as calcium, potassium, or sodium. -Not as efficient at increasing blood ketone levels. -Greater gastrointestinal distress -Potential counterindication in people living with hypertension. ≈0.4mmol-1.0mmol/L BHB Ketone monoester -(R)-3-hydroxybutyl (R)-3-hydroxybutyrate) formed by the synthesis of R-Butanediol and R-βHB -Has shown the highest, fastest, and longest lasting increases in circulating ketones -Intense bitter flavor. -Up to 3-6.5mmol/L BHB at well tolerated doses of 573mg/kg. 1-3 butanediol -Precursor of BHB that follows a series of short oxidative reactions of alcohol metabolism ending with the oxidation of β-hydroxybutyraldehyde to BHB. -Usually found in commercial presentations bonded within the ketone monoester molecule to potentialize its effects. -0.5-1.0mmol/L at well tolerated doses of 35-50g Medium chain fatty acids / medium chain triglycerides -These molecules are broken down by lipases in the gut and transported to the liver to undergo beta oxidation into ketone bodies. -Capability to increase ketonaemia dependent upon nutritional state and (other) energy sources availability. -Depending on general metabolic and nutritional state, can range from 0.5-1.0mmol/L with moderately tolerated doses of 30g Despite their growing availability, ketone supplements are currently marketed mostly based on claims that are not supported by scientific research. Overstretched claims that are not backed by strong scientific study include suggestions for increased athletic performance, restoration of metabolism (which is non-specific and difficult to link to an outcome measure), increased focus and cognition, and other bold statements with little to no supportive evidence. However, escaping from marketing and business strategies and citing the work from our laboratory’s colleagues: “Exogenous 13 ketone supplementation [is] an emerging tool for physiologists with potential as a metabolic therapy” (19). Since their conception, exogenous ketone supplementation has seen multiple commercial advances. Synthesizing and commercializing pure ketone bodies is expensive and the pharmacokinetic profile reveals their inadequacy at maintaining elevated blood ketone levels for more than a few hours. Therefore, commercial forms of ketone supplements are evolving towards the concept of incorporating ketogenic precursors, which can be converted (typically by the liver) to ketone bodies to gain a more sustained elevation of blood ketones (30). Given the nature of the initial goals in exogenous ketone research, an important proportion of the following research has been related to human performance, and, more specifically, sports performance (31). Some research investigating exogenous ketones in the context of athletic performance found that exogenous ketosis consistently lowered blood glucose at different doses. In 2022, our laboratory published a systematic review and meta-analysis describing the effects of exogenous ketone supplements on blood glucose. The results of this analysis were consistent with the following conclusions: (32) • Both ketone monoesters and ketone salts successfully increased circulating β-HB levels to a mean of 1.73 mmol/l, whilst decreasing glycemia by -0.54 mmol/l. • Ketone monoesters have a significantly greater impact than ketone salts at increasing β-HB levels and consequently decreasing blood glucose levels. • There is a wide spread of the prevalence of side effects, ranging from <1% of doses causing side effects to 70% of participants experiencing any symptoms, which tended to be gastrointestinal in nature, including nausea, bloating, abdominal pain, vomiting, heart burn, and other gastrointestinal distress. 14 • Limitations of the analysis and the studies related to most studies being single dose trials, a lack of long-term studies, potential conflicts of interest as some of the authors have affiliations with the commercial aspect of ketone supplements, as well as heterogeneity within participants, doses, and study designs. The glucose-lowering effect of exogenous ketone supplements represents a potential new strategy in the treatment of T2D. Within work published by our laboratory, a direct assessment of the effect that a single dose (482 mg/kg body mass) of ketone monoester, as compared to non-caloric placebo, taken 30 minutes prior to a standardized oral glucose tolerance test (OGTT) showed that β-HB levels rose up to a mean of 3.2mmol/l and glucose area under the curve (AUC) following the OGTT decreased by -17%. Interesting, there were no apparent effects on insulin production during the OGTT, suggesting the exogenous ketone supplement may have improved insulin sensitivity (although this was not directly measured) (33). Using a similar study design, a single dose of ketone monoester (same dose as previous mentioned trial: 482 mg/kg body mass) conducted in people living with obesity showed a reduction of -11% in glucose in response to an OGTT (34). This was accompanied by a -21% reduction in non-esterified fatty acids in the ketone monoester compared to placebo condition, suggesting a potential impact on lipolysis and lipotoxicity (25). More recently, our lab conducted a randomized, placebo-controlled crossover trial comparing 14 days of pre-meal (three times per day) ketone monoester vs placebo on free-living glycemic control assessed by continuous glucose monitoring (CGM). CGM analyses revealed that ketone monoester supplementation resulted in an 8% lower postprandial glucose and 7.8% lower 24 hour average glucose levels in people living with obesity. In addition, brachial artery flow mediated dilation, which is a non-invasive technique to assess vascular endothelial function, increased after the 14-day supplementation with exogenous ketones, suggesting potential benefits of exogenous ketones on cardiovascular health (35). Outside our research group, a single arm trial evaluated the tolerability 15 and effect of the ketone monoester supplement on blood glucose in patients with type 2 diabetes during a 4-week period. Overall, the ketone monoester supplementation was well tolerated and showed improved (lower) CGM glucose metrics when compared to baseline readings (36). This 28-day trial is, to our knowledge, the longest trial of exogenous ketone supplementation in people living with T2D although its single arm nature and lack of any dietary reporting makes any firm conclusions on the impact of exogenous ketones on glucose regulation tenuous and preliminary. Our lab recently published a 14-day randomized double-blind, placebo-controlled crossover trial of ketone monoester supplementation in people living with T2D and, although the supplement appeared well-tolerated in the short-term, there was no evidence for glucose-lowering assessed by plasma fructosamine (which reflects average blood glucose over ~14 days) in this higher-quality trial. These results suggest that longer term supplementation may be needed to see any influence of exogenous ketones on markers of glucose control. Exploring feasibility In the context of health and upon the development and proposal of any potential intervention (lifestyle, medications and/or supplementation), pilot and/or feasibility studies are important. The terms pilot and feasibility have often been used interchangeably by researchers and it is even debatable whether a pilot trial is embodied within a feasibility study, or the feasibility study is embodied within the pilot trial (37). In general terms, a pilot trial would try to pre-model the methodology for a later bigger trial, taking the advantage of the smaller sample size to further assess the quality of the study design and the precision of outcome measures, as well to estimate potential intervention’s effect size and, with it, support a further design’s sample size and expected results. As for feasibility trials, researchers will use them to evaluate potential drawbacks that can preclude successful implementation of a bigger study (38). These drawbacks can be design related, intervention related, or individual related. Considering this, it is possible to design a study that can 16 accomplish multiple goals, and hence the terms pilot trial and feasibility study can be understood as a compound in many cases. Pilot and feasibility studies will be considered within exploratory approaches, hence, it is difficult to define exact sample size goals, success rates or successful outcome measures’ results, and, whether or not formal statistical analyses (or hypothesis-testing) are conducted. In general, pilot and feasibility studies will report descriptive data and emphasize researchers’ interpretation to synthesize key findings (39). As mentioned above, exogenous ketone supplementation has shown adequate tolerance and safety in early studies, although many are short-term (single dose and up to 28 days) (36). The existing studies and trials performed in clinical and non-clinical environments suggest that it is indeed possible for people to comply with daily consumption of exogenous ketone supplements, at least in the context of a scientific research study. However, tolerability and experimental trials have repeatedly reported specific side effects related to consumption, which include bitter taste, headache, nausea, and general gastrointestinal distress (32). These side effects are usually mild (but present) and, thus, studies conclude positive tolerance of ketone supplementation. Further, despite not being adequately captured in many studies, it is widely known that the extremely bitter (and unpleasant) taste of the ketone monoester supplement would be a barrier to regular, long-term consumption. Being that T2D is a disease that in most cases will require medical treatment for multiple years or even for life (40), considering exogenous ketone supplementation as an aid to control glycemic levels in this patient population will require ketone supplements to be well tolerated over a longer term. Given the promise of exogenous ketones for lowering blood glucose, the potential concerns around taste and gastrointestinal distress, and the paucity of longer-term studies, we identified the need for a prolonged feasibility trial to explore participant recruitment, retention, compliance/adherence, experiences, the utility of different assessment tools, and preliminary efficacy to advance research on exogenous ketones and T2D. The ultimate goals of a 17 feasibility trial would be to inform a potential future, more definitive randomized controlled trial (RCT), where the appropriate methods could be used to test whether exogenous ketones might represent a novel treatment strategy for T2D. Given that a potential future efficacy RCT would likely require a large sample size. Testing the feasibility of a remote trial design like this could facilitate the future conduct of a remote RCT with a broad recruitment area from a central site. 18 Objectives: Primary Objective: To determine the feasibility of a randomized controlled trial (RCT) testing the effects of prolonged consumption of a ketone supplement drink in adults with type 2 diabetes in a free-living environment for 90 days, as determined by: i. Recruitment rate of eligible participants into the trial. ii. Compliance as measured by the self-reported volume of ketone supplement drink consumed. iii. Retention as measured by the number of participants that complete the study. Criteria for Determining Feasibility (Primary Outcome): i. A recruitment rate of at least 4 participants per month (which will ensure the study is fully enrolled within a 5-month timeline); ii. ≥67% of the total drinks provided being consumed by participants as determined via self-report (i.e., an average of two out of three drinks per day being consumed); iii. ≤30% of recruited participants withdrawing from the study. Secondary Objectives: i. To estimate assessment tools and capability to successfully retrieve all desired outcome data and evaluate potential burdens and solutions for a definitive RCT. ii. To estimate the preliminary effects of a ketone supplement to help estimate the required sample size for the definitive RCT for potential outcome measures. Main outcome measures are: 19 a. Measures of glycemic control i. HbA1c assessed before and after the 90-day trial. ii. Average glucose, estimated glycosylated hemoglobin, blood glucose area under the curve, low blood glucose index, high blood glucose index, and mean amplitude of glycemic excursions assessed by continuous glucose monitoring during a 5-day baseline period followed by the first 9 days of intervention and during the last 14 days of the study. b. Supplement acceptability assessed via questionnaires at the beginning and at the end of the trial. c. Gastrointestinal distress assessed via questionnaires at the beginning and at the end of the trial. d. Self-reported body weight and waist circumference assessed at the beginning and at the end of the trial. e. Self-reported energy intake assessed via dietary 24-hour recalls at the beginning and at the end of the trial. f. Levels of perceived hunger assessed via questionnaires at the beginning and at the end of the trial. g. Food cravings assessed via questionnaire at the beginning and end of the trial. h. Self-rated health and its impact on everyday life assessed via questionnaire at the beginning and at the end of the trial. i. Markers of inflammation assessed at the beginning and at the end of the trial. 20 j. Systolic and diastolic blood pressure assessed at the beginning and end of the trial. k. Overall supplementation and intervention acceptability assessed by means of an open-ended questionnaire at the end of the trial and/or in case of withdrawal from the trial. iii. To evaluate the acceptability of the intervention and data collection procedures to participants, assessing the feasibility of implementing the intervention. 21 Methods Ethical approval: The present study was approved by the University of British Columbia Clinical Research Ethics Board (UBC CREB number: H22-00644). The study conformed to the standards set by the Declaration of Helsinki, and was pre-registered (NCT05983562). Every participant signed a digital informed consent form after online screening. Sample size: As a pilot trial, looking to assess prolonged effects of ketone supplementation on patients with T2D, a total sample of N=20 (10 participants per group) was deemed sufficient to explore feasibility and set the basis for future broader studies. Inclusion criteria: To be eligible for this study participants had to be: i. diagnosed with type 2 diabetes by a physician at least 1 year prior, ii. between 30-69 years of age, iii. stable use of glucose-lowering medications for at least three months. Exclusion criteria: Participants were not eligible for this trial if they were/had: i. trained endurance athletes, ii. actively attempting to gain or lose weight, iii. a history of mental illness or existing neurological disease, 22 iv. a history of cardiovascular events in the last two years, v. hypoglycemia, vi. irritable bowel syndrome, or inflammatory bowel disease, vii. taking SGLT2 inhibitors or insulin, viii. using more than 2 classes of glucose-lowering medication, ix. following a ketogenic diet or regularly taking ketone supplements, x. unable to commit to a 95-day trial, xi. unable to follow remote guidance by internet or smartphone, xii. taking natural or over-the-counter supplements specifically designed to lower blood glucose (e.g., berberine, bitter melon). Assessment tools: The primary outcome measure was feasibility. Secondary outcome measures included glycemic control, supplement acceptability, gastrointestinal distress, body weight, dietary intake, as well as hunger and satiety. Acceptability of the intervention and data collection procedures were also assessed. • Laboratory blood sample analysis: Venous blood samples were collected at an accredited clinical laboratory close to the participant’s place of residence before and after ketone supplementation. HbA1C is the main clinical marker of glucose control in T2D and is used by healthcare practitioners to guide T2D medication prescription. HbA1c measured the level of glucose bound to hemoglobin within red blood cells giving a general reflection of the average concentration of blood glucose for the past 2-3 months. Lipid profile (being total cholesterol (TC), triacylglycerol (TAG), high-density lipoproteins (HDL), non-high-density lipoproteins (nonHDL), low-density lipoproteins (LDL)) was also assessed given the 23 association between T2D and dyslipidemia and the potential for ketones to impact lipid metabolism (34). • Continuous glucose monitor (CGM): CGM measures glucose in the interstitial fluid with proprietary manufacturer algorithms converting interstitial glucose to an estimation of blood glucose concentration. We used the FreeStyle Libre 2 monitor which is factory-calibrated, thereby reducing participant burden. This device can be worn for up to 14 days and records glucose every 15 minutes and therefore can detect trends and patterns of hyperglycemia and hypoglycemia across the day. Key outcome measures included 24-hour average glucose, glycemic area under the curve (AUC), and glycemic variability. CGM data were collected at three distinct timepoints in this study: for 5 days prior to the start of supplementation (baseline), during the first 9 days of supplementation (active 1), and during the last 14 days of supplementation (active 2). • Anthropomorphic measurements and cardiovascular indicators: waist circumference was measured and self-reported by the participant using a measuring tape provided in the study kit and following directions from the researchers. Blood pressure was measured by the participant using a blood pressure monitoring device provided in the study kit and following directions from the researchers. These measures were taken as general markers of cardiometabolic health and to explore potential changes in body composition as ketones have been implicated in appetite regulation (41) and cardiovascular risk (42). • Questionnaire assessments: I. Health screening questionnaire A customized Medical and Health Status Questionnaire was used to assess inclusion/exclusion criteria and prior history of medical conditions at baseline (Appendix A). 24 II. Weekly compliance questionnaire: Participants were asked to indicate how many drinks they consumed over the course of a week via a weekly REDCap survey to measure compliance. Weekly compliance questionnaire consisted of a single question being: How many of the drinks did you consume over the last 7 days? Participants were indicated to respond within 0-21 accordingly. III. Theory of planned behavior questionnaire: A customized research questionnaire was used to assess likability of participants to comply/adhere to intervention indications. Participants were asked to answer this questionnaire at the beginning and at the end of the intervention (Appendix B). IV. Drink acceptability questionnaire: Acceptability of the drink (ease of compliance, taste, etc.) was assessed via questionnaire at day 1 (after first week of supplementation) and by the end of the intervention (Day 90). A 7-point Likert scale was used (Appendix C). V. Godin physical activity questionnaire: A validated physical activity survey was used to assess any changes in typical activity at the beginning and at the end of the trial (43) (Appendix D). VI. EuroQol 5D-5L (EQ 5D-5L): Standardized survey to determine general health status and used to detect any potential changes throughout the trial, assessed at the beginning and at the end of the trial (44)(Appendix E). VII. Food cravings questionnaire: 25 The Food Cravings Questionnaire – Short version was used to detect an increase or decrease in participants general food cravings at the beginning and at the end of the trial (45) (Appendix F). VIII. Visual Analog Scale (VAS) Hunger and fullness: VAS questionnaire applied to detect any potential interactions between the supplementation condition and participant’s appetite perception at the beginning and at the end of the trial, this was done concatenated to the rest of the questionnaire assessments stated in this section (46)(Appendix G). IX. Diet assessment questionnaire: Participants completed food logs using the Automated Self-Administered 24-Hour (ASA24®) Dietary Assessment Tool. This is a web-based tool which requires a log-on and password. All participants were registered by the researchers using a dummy account linked only to the participant identificatory number and no personal information. Participants were instructed and reminded to fill in this assessment before the start of the trial for inclusion and at the end of the intervention (day 90). X. Open-ended acceptability questionnaire: An open-ended feedback questionnaire was administered at the end of trial or in case of withdrawal to better asses the acceptability and/or rejection of the intervention (e.g., feedback on supplement, including dose, overall taste, overall opinion, and future attitude towards ketone supplements) (Appendix H). 26 Study flow: Product selection and dose prescription Careful consideration by the trial team, rooted in extensive experience conducting studies using exogenous ketones, informed the selection of the exogenous ketone supplement for this trial. As mentioned previously, despite the most evidence of efficacy (primarily in single dose/visit studies), the ketone monoester supplement has a significant drawback in terms of taste. Feedback from participants in previous 14-day studies reduced confidence in the ability of participants to consume this supplement thrice daily for 90 days in this trial. To address this issue, our laboratory colleagues designed and published in 2023 a simple acute trial comparing the ketone monoester supplement with two related commercially available ketone supplements (47). The first comparator supplement was Kenetik®, which is free R-β-hydroxybutyric acid combined with R-1,3-butanediol at a 1:1 ratio – essentially representing the same molecules as the ketone monoester but not bound together within an ester bond. The second comparator was KetoneIQ®, which contains R-1,3-butanediol, the ketone precursor constituent in the ketone monoester. This double-blind randomized cross-over pilot study demonstrated that a pragmatic approach with a consistent dose of 10 g of each active ingredient can raise blood BHB above 1 mmol/L, peaking ~60 minutes after supplement consumption. Similarly, each supplement was able to lower blood glucose concentration with the glucose nadir mirroring the time of highest BHB concentration. The Kenetik® product (free R-β-hydroxybutyric acid combined with R-1,3-butanediol at a 1:1 ratio) emerged as the ketone supplement with the highest taste ratings and acceptability amongst participants. Based on this favorable metabolic response and taste/acceptability profile, we set Kenetik® at 10 g of active ingredient per dose as the experimental supplement for our trial. Further trials have been done in the specific context of type 2 diabetes and aiming for blood glucose lowering effect as the main outcome. These trials have consistently replicated this effect when using individualized doses up to 27 0.5g/kg of active compound which for the mean human body weight would range around ≈30-50g per dose (48). However, leaning on the fact of this trial’s main outcome is not to test efficacy but to assess feasibility, and knowing that the previously mentioned study also showed a tendency to lower blood glucose at standard doses, we decided to prescribe a dose similar to the usual suggested dose of these commercial brands. For prescription we opted for 3 daily doses of 10 g of active ingredient per day, which we encouraged, though not mandated, participants to consume before every big meal (breakfast, lunch, and dinner) given the glucose-lowering impact of pre-meal ketone supplementation demonstrated in previous studies (33) (49) (50). The 10 g dose is the recommended single serving dose by the company and that which has been tested within laboratory studies demonstrating tolerability and without causing major side effects (46). Supplement details Kenetik drink was developed by VitaNav Laboratories and launched to market in 2021. Its base is a beverage flavored with allulose, stevia, and natural flavors and its formula contains a 5 g dose of R-β-hydroxybutyric acid with a 5 g dose of R-1,3-butanediol, in 59ml of concentrated drink. We directly contacted the company’s CEO who agreed to sell the product for research purposes. Although the exact energy content of exogenous βHB and 1,3-butanediol is not abundantly clear, the stated calorie content from the product monograph of each dose is 60 kcal (based on 6 kcal / g βHB). The supplement company also created a flavor and image-emulated product containing the same sweeteners and natural flavors with no caloric content; drinks were differentiated with a different lot number with identity blinded to the research team. As Kenetik is a US-based company, we hired a third-party company to arrange importation and shipping logistics to our university’s campus. 28 Shipment and delivery to participants To deliver product materials to participants, we used the international shipping company FedEx ground services. Foreseeing any potential eventualities and seeking to economize and maintain a pragmatic approach that could be replicable for future and bigger trials, we arranged two instalments per participant where we delivered all the material that would be needed for the first and second half of the trial. The first shipment was made after the participant was randomized and the second one on day 40 of the trial (trial flow detailed in Figure 3 below). Each shipment consisted of a 40 x 30 x 15 cm box, which took 1-2 business days to be delivered to the participant’s home. Recruitment and randomization For recruitment intentions, we focused our efforts on the province of British Columbia (BC), first contacting participants from previous trials from our laboratory that might be interested in participating (an ethics pre-approved script email was sent), adding newspaper and campus advertisements (also approved by the REB and campus guidelines), and introduced a third-party recruitment company (Wayturn) to manage social media advertisements and pre-screen and filter potential participants. In every case, we screened each participant individually via a phone call made by one of the researchers involved in this study, following a prewritten script to ensure no information was omitted (email and phone scripts included in supplemental material). After ensuring eligibility, an informed consent RedCap link was sent via email (available in supplemental material). Once the informed consent was signed, potential participants were assigned a participant ID and were sent via email initial survey assessments, a Valley Medical Valley Labs (Kelowna) or LifeLabs (Vancouver and rest of BC) blood requisition blinded with participant ID, and a preassigned ID and password for the Automated Self-Administered 24-hour Dietary Assessment Tool (ASA-24), which they were asked to fill in once at baseline. At the completion of these three items and upon receipt 29 of the laboratory blood work results at our office’s fax, participants were randomized by a researcher inputting the participant ID into RedCAP and receiving the group allocation according to a randomization schedule uploaded by a third-party researcher and officially included in the study. A flow chart for participant recruitment is shown in Figure 3. Figure 3. Participant recruitment flow chart for randomization. 30 Study design: Once randomized, participants were sent a first installment of ketone supplements (135 bottles, for the first 45 days of the trial) and a starter kit including: instruction booklet (available in supplemental materials), measuring tape, blood pressure monitor, and one continuous glucose monitor. Upon reception of this material by the participant, a video call meeting was scheduled with one of the researchers. In this call, the researcher and the participant went together through the instruction booklet and material, the flow of the study was explained, and researchers made sure that every aspect was clear and understandable for participants. They were also reminded that their participation was voluntary and that they were free to withdraw at any point if they felt the need to for any reason. Participants were encouraged to share their concerns with researchers at any moment during the study. Finally, a start date for the trial was set together, and, if participants decided to start right away, they were offered the possibility to insert the CGM with remote guidance from the researcher. Once the start date was set, the study timeline overview was the following: Study timeline overview Participants were guided to follow this study timeline for a total of 95 days: 1. Day -5: set as start day, the participant inserted the CGM. Called “day -5” as this is prior to intervention. The information obtained from these 5 days of CGM data was taken as a baseline measured to be compared with the one obtained at the beginning of supplementation (step 5 below) to explore acute/short-term changes that might suggest impact on glucose control and at the end of the study (step 8 below) to explore longer-term effects. 31 2. Day 0: participants 1) took and sent via email waist circumference and blood pressure readings and 2) answered medical history questionnaire, GODIN physical activity questionnaire, Theory of Planned Behaviour questionnaire, Cravings questionnaire, Hunger/fullness questionnaire, and ASA-24 dietary assessment. 3. Day 1: 1) participants began supplement consumption every day, three times a day, ideally before each meal, 2) answered product acceptability questionnaire and gastrointestinal distress questionnaire. 4. Day 7: participants were asked to record their weekly compliance, from day 7 onwards, via weekly RedCap email survey which asked them to self-report the number of supplements taken within the last 7 days. 5. Day 9: participants removed 1st CGM. 6. Day 40: researchers sent via FedEx delivery service the second instalment of supplements (135 bottles, for the remaining 45 days of the trial) plus a second CGM reader for the last 14 days of the trial. 7. Day 77: participants were asked to insert second CGM. 8. Day 90: participants removed second CGM. Participant were sent an email asking to 1) take and send waist circumference and blood pressure readings, 2) answer all questionnaires as above 3) get follow-up blood work done within the next 7 days at their local laboratory (Medical Valley labs or LifeLabs). End of trial: study trial was successfully concluded once the second laboratory results are received at our office’s fax. Figure 4, included in the instruction booklet that participants received, graphically describes the study flow. 32 Figure 4. From participant’s instruction booklet in the supplemental materials, graphically represents the study flow in an understandable way for participants to be able to follow and refer when needed throughout their participation. Data management and participant security: All data was de-identified, stored within REDCap or Open Science Framework (OSF) folders with restricted access, and only accessible by authorized study team members. Upon randomization, a unique ID number was established containing no personal information. Questionnaire distribution 33 was done via REDCap, a researcher developed tool for compliant contact between participants and researchers. All data was be stored in OSF on Canada-based servers. No participant information was kept on personal computers. Laboratory requisitions were elaborated as per first name and personal ID number previously established for the participant, and the results were sent directly via fax to the Exercise and Inflammation Laboratory located at the University of British Columbia Okanagan Arts building, only members of the laboratory have access to the room. Statistical Analyses Descriptive characteristics and simple outcome variables were summarized as means and standard deviations or simple counts. Given this was a pilot trial and, by definition, not powered for detecting significant differences in outcome variables, p-values are not reported, and no interpretation of statistical significance is provided. It was initially planned to conduct preliminary statistical analyses using linear mixed models with group and time as fixed factors and participant ID as a random factor to derive effect estimates with 95% confidence intervals for the within group and between group differences. However, due to higher-than-expected drop-outs in the ketone group and higher than expected missing data, these statistical models could not be used due to violation of minimal data and/or poor model fits. As such, mean and standard deviation for efficacy outcomes at each timepoint are presented for all available participants (analogous to intention-to-treat analyses, with the caveat that each timepoint is made up by a different number/group of participants) and for only participants with complete data (analogous to per-protocol analyses, with the caveat of low sample size) as tables with limited interpretation of efficacy. 34 Results A total of 38 people were screened for eligibility. Five of them were contacted via our laboratory’s participant pool from previous studies, two leads were referred by ongoing enrolled participants in the present trial, three more leads were self-referred to us directed by local newspaper advertisements, and the remaining 28 participants were referred by third party recruitment company WayTurn. It is important to mention that the recruitment company proved efficient at sending referrals to our trial being able to refer 33 leads from two separate social media advertising campaigns, both of which lasted approximately 15 days. However, from these referrals, we were unable to contact 5 of them (hence not considered within the screened participants), upon further screening 11 leads did not continue with the process, and 1 of them was ineligible according to our exclusion criteria (active smoking). Summing up, from the 33 leads referred by WayTurn, we were able to enroll 17 participants in the trial. From the total pool of screened participants, 10 leads did not continue with the enrollment process due to potential lack of interest, 2 screened participants actively refused to wear a CGM which was required at two timepoints during the trial, and 4 people were not included according to exclusion criteria being incompatible medication (n = 2), active smoking (n = 1), and incompatible supplementation (n = 1). After screening for eligibility, 22 participants signed the online informed consent but two of them did not continue with the process. A total of 20 participants were randomized and enrolled in the trial (n=10 female and n=10 male), 10 of which were allocated to the experimental group (n=4 female and n=6 male) and 10 allocated to the placebo/control group (n=6 female and n=4 male), sample characteristics plus baseline and follow up anthropometrics and blood pressure records are furtherly described in table 2. Recruitment began in June 2023 with the first participant being enrolled June 29th, 2023 and the 35 last participant enrolling December 11th, 2023, completing screening and enrolment in 6 months with a mean recruitment rate of 3.3 participants per month. Mean participant age was 53.5 ± 7.1 years old for the total of the sample and 53.9 ± 7.5 years old and 53.1 ± 7.4 years old for the experimental and control group, respectively. Of the 20 participants, 15 of them completed the trial successfully, which was considered as not actively requesting to withdraw from it. Of the 5 withdrawals, 4 participants had been allocated to the experimental group, whilst 1 was in the control group. Withdrawal was due to different levels of gastrointestinal distress (n=2) and urge to vomit (n=2) in the experimental group and dislike of drink flavor (n=1) from the control group. The consort flow chart is described in Figure 5. 36 Figure 5. Consort flow chart for participant recruitment, screening, enrollment, allocation, and trial completion/withdrawal. 37 Table 2. Sample characteristics and cardiovascular and anthropometric records. Sample characteristics, cardiovascular and anthropometric records Total Ketone Placebo N 20 10 10 Age, years 53.5 ± 7.1 53.9 ±7.1 53.1 ± 7.4 Female, N (%) 10 (50) 4 (40) 6 (60) Male, N (%) 10 (50) 6 (60) 4 (40) BASELINE Total Ketone Placebo Systolic BP (mmHg) 121 ± 8 123 ± 8 120 ± 9 Diastolic BP (mmHg) 80 ± 7 79 ± 8 80 ± 7 Waist circumference (cm) 108.5 ± 15.5 112.8 ± 18.8 104.7 ± 11.7 FOLLOW-UP Systolic BP (mmHg) 121 ± 11 124 ± 12 120 ± 10 Diastolic BP (mmHg) 78 ± 8 77 ± 11 78 ± 5 Waist circumference (cm) 102.4 ± 15.0 105.2 ± 19.8 100.1 ± 11.2 Data presented as mean ± standard deviation. Supplementation compliance The results for weekly supplement consumption compliance are shown in figure 6. In week 1, for the ketone group mean compliance was 20.5 ± 1.1 out of 21 (98%) and for the placebo group mean compliance for week was 18.5 ± 4.6 (88%). Week 13 (last week of trial) consisted of a total of 6 days; hence the maximum number of bottles consumed was 18. For the ketone group, compliance decreased compared to week 1 with a compliance percentage of 81.5% (out of the 18 bottles). In the placebo group, last week’s compliance percentage was 67.4%. 38 Figure 6. Time course showing the compliance expressed by patients with Type 2 diabetes (T2D) administered orally thrice a day for 90 days with an exogenous ketone supplement and a taste-matched placebo. Values are means  SD. Week 13 adjusted for compliance percentage as maximum number of supplement bottles consumed was 18. Secondary outcome completion rate Table 3 describes the rate of completion rate for secondary assessments and is further described here: • Blood work: All participants completed blood work at the baseline timepoint. Follow-up completion rate went down to 80%. Two participants (n=2) were unable to their blood draw done at their local laboratory for personal reasons, a third participant commented he did go to his local laboratory, but the staff refused to register their requisition as per his participant ID and registered his personal information instead. Hence the results were not sent to our laboratory’s fax and researchers were unable to retrieve this information. 0.005.0010.0015.0020.0025.001 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3COMPLIANCE (# OF BOTTLED SUPPLEMENTS TAKEN)TIME (WEEK OF INTERVENTION)Ketone Placebo39 • Anthropometrics: One participant (n=1) did not measure their waist circumference in any way for the baseline timepoint without giving any explicit reason. A different participant (n=1) failed to provide this information at the follow up timepoint. • Cardiovascular markers: 17 out of 20 participants provided 3 consecutive blood pressure reading at baseline and 14 out of 15 participants did so at follow up timepoints. • ASA 24 dietary intake assessment: At baseline 19 participants completed this assessment and at follow up 14 did. No explicit reason was given by any participant that failed to fulfill the dietary intake assessment. • Continuous glucose monitoring: Completion rate for this outcome was 80% for both baseline and follow up timepoints. At baseline, two participants withdrew within the first week of the trial, hence, the device was not worn for the full time point; one participant had compatibility issues with their mobile device and the CGM. A fourth participant’s CGM failed within the first days after insertion. Similar situations took place at the follow up timepoint; the same (n=1) participant had compatibility issues, another participant’s CGM failed within the first days, and a third participant did not insert the CGM when indicated. • General health, physical activity surveys, and drink acceptability and gastrointestinal distress surveys: Completion rate for these REDCap questionnaires was 100% at the baseline timepoint and 93% for the follow up timepoint, with the exception of the gastrointestinal distress questionnaire (for which the link was delivered separately, owing to the participant needing to try the supplement first) being completed 80% of the time at baseline. 40 Table 3. Participants’ rate of compliance/outcome data availability for the study divided by timepoint. Expected sample and percentage as per enrolled participants at each timepoint. Baseline (N=20) Post (n=15) Blood work 20 (100%) 12 (80%) Anthropometrics (waist circumference) 19 (95%) 14 (93%) Cardiovascular markers (blood pressure) 17 (85%) 14 (93%) ASA 24 dietary assessment 19 (95%) 14 (93%) CGM 16 (80%) 12 (80%) Theory of planned behavior 20 (100%) 14 (93%) EQ 5D 5L 20 (100%) 14 (93%) VAS of appetite 20 (100%) 14 (93%) Godin physical activity 20 (100%) 14 (93%) Food cravings survey 20 (100%) 14 (93%) Drink acceptability Likert scale 16 (80%) 14 (93%) Gastrointestinal distress survey 17 (85%) 14 (93%) Data presented as total number of records per category and percentage (%) of compliance according to enrolled participants per time point. Preliminary efficacy Results for the preliminary efficacy of each outcome measure are reported in Tables 4-16. Each table has outcomes reported as mean and SD, for the full available data (intention-to-treat) and for only paired results (complete data, per protocol) at both baseline and follow up timepoints Related to laboratory blood work, we noticed a mean decrease from 8.0% ± 2.4 to 6.0% ± 0.8 (intention-to-treat data) or from 7.2% ± 2.6 to 6.0% ± 0.8 (paired data) for Hb A1c within the ketone group when comparing baseline to follow up timepoints (Table 4). This same tendency for lowered 41 blood glucose can be seen in Table 16, which presents CGM data wherein both intention-to-treat and paired data reveals directional decreases in 1) average glucose, 2) estimated Hb A1c, and 3) High blood glycemic index when comparing baseline and follow up timepoints in the ketone group. There were no trends seen for changes in markers of glycemic control in the placebo group. Pairing data proved to be useful in several cases where it appeared there was a trend for group differences (or potential time X condition interactions) in intention to treat results. When data were paired, these apparent trends were either reduced in magnitude, disappeared, or even reversed, letting us understand that this may be a potential artifact created by the reduced sample size and data availability. Table 6 shows mean results for waist circumference readings at baseline and follow up timepoints, with intention-to-treat data potentially overestimating or creating a trend to decrease this metabolic marker when comparing baseline and follow up timepoints in the ketone group. However, paired data minimizes the potential interpretation of this being a trend, which is due to a participant (or participants) with higher-than-average waist circumference only included in the baseline but not follow-up data. In the same way, paired data was also useful to detect potential trends that are not shown by intention-to-treat data. This can be seen in Table 14 where the mean results for the Visual Analog Scale of gastrointestinal distress are shown. Paired data shows that, within the participants that successfully completed the trial and answered this assessment tool at both baseline and follow up time points, there was an increase in mean gastrointestinal distress scores in the ketone group whereas the placebo group appeared to stay constant. 42 Table 4. Preliminary effect produced by the consumption of thrice a day for 90 days exogenous ketone supplement compared to a non-caloric taste matched placebo in people living with T2D showing baseline and follow-up readings for blood chemistry levels focused for lipidic profile. Ketone Placebo Ketone Placebo Available data Paired data HbA1c (%) Baseline 8.0 ± 2.4 (10) 6.5 ± 0.7 (10) 7.2 ± 2.6 (5) 6.5 ± 0.5 (5) Follow up 6.0 ± 0.8 (5) 6.4 ± 0.3 (7) 6.0 ± 0.8 (7) 6.4 ± 0.3 (7) AST (U/L) Baseline 28.4 ± 17.6 (10) 30.5 ± 15.6 (10) 22 ± 10.4 (5) 29 ± 16.9 (7) Follow up 25.2 ± 8.1 (5) 31 ± 16.1 (7) 25.2 ± 8.1 (5) 31 ± 16.1 (7) ALT (U/L) Baseline 32.3 ± 16.6 (10) 43.4 ± 39.6 (10) 23.6 ± 14.7 (5) 41.9 ± 41.9 (7) Follow up 23.4 ± 9.3 (5) 40.1 ± 39.2 (7) 23.4 ± 9.3 (5) 40.1 ± 39.2 (7) TC (mmol/l) Baseline 4.85 ± 1.1 (10) 5.6 ± 1.4 (10) 4.8 ± 0.7 (3) 5.0 ± 1.4 (4) Follow up 5.0 ± 1.9 (5) 5.7 ± 1.1 (7) 4.7 ± 1.8 (3) 5.3 ± 1.4 (4) TAG (mmol/l) Baseline 2.3 ± 1.5 (9) 3.1 ± 2.5 (10) 1.3 ± 0.7 (3) 1.6 ± 0.7(4) Follow up 2.1 ± 2.5 (5) 3.2 ± 2.2 (7) 1.1 ± 0.5 (3) 1.6 ± 0.7 (4) HDL (mmol/l) Baseline 1.2 ± 0.4 (10) 1.2 ± 0.4 (10) 1.7 ± 0.3 (3) 1.2 ± 0.4 (4) Follow up 1.3 ± 0.5 (5) 1.1 ± 0.3 (7) 1.4 ± 0.6 (3) 1.2 ± 0.3 (4) Non-HDL (mmol/l) Baseline 3.6 ± 1.2 (9) 4.3 ± 1.5 (9) 3.1 ± 0.4 (3) 3.9 ± 1.2 (4) Follow up 3.7 ± 1.8 (5) 4.5 ± 1.2 (6) 3.3 ± 1.2 (3) 4.1 ± 1.3 (4) LDL (mmol/l) Baseline 4.35 ± 3.6 (7) 4.2 ± 3.5 (8) 2.5 ± 0.6 (3) 3.1 ± 1.0 (4) Follow up 2.6 ± 1.2 (4) 3.4 ± 1.0 (4) 2.8 ± 1.3 (3) 3.4 ± 1.0 (4) Data presented as mean ± standard deviation and (n) sample size per outcome and time point. Left columns show results for all data available, right columns show results for paired data at both time points. 43 Table 5. Preliminary effect produced by the consumption of thrice a day for 90 days exogenous ketone supplement compared to a non-caloric taste matched placebo in people living with T2D showing baseline and follow-up readings for blood cell count and inflammatory markers. Ketone (10, 5) Placebo (10, 7) Ketone (5) Placebo (7) HGB (g/L) Baseline 144 ± 12 143 ± 12 142 ± 13 142 ± 14 Follow up 141 ± 11 122 ± 54 141 ± 11 122 ± 54 WBC (cells 10*9/L) Baseline 6.7 ± 1.5 6.7 ± 2.6 6.8 ± 1.7 7.2 ± 2.9 Follow up 7.0 ± 1.7 7.0 ± 2.3 7.0 ± 1.7 7.0 ± 2.3 Platelets (cells 10*9/L) Baseline 239 ± 42 251 ± 80 241 ± 42 253 ± 91 Follow up 261 ± 53 259 ± 112 261 ± 53 259 ± 112 RBC (cells 10*12/L) Baseline 5.63 ± 2.76 4.80 ± 0.27 4.65 ± 0.38 4.88 ± 0.26 Follow up 4.69 ± 0.30 4.91 ± 0.37 4.69 ± 0.30 4.91 ± 0.37 HCT (L/L) Baseline 0.43 ± 0.03 0.42 ± 0.02 0.42 ± 0.03 0.42 ± 0.03 Follow up 0.41 ± 0.02 0.3 ± .03 0.41 ± .02 0.43 ± 0.03 MCV (fL) Baseline 88 ± 3 87 ± 3 89 ± 2 85 ± 1 Follow up 89 ± 3 86 ± 2 89 ± 3 86 ± 2 MCH (pg) Baseline 29.9 ± 1.4 29.8 ± 1.6 30.4 ± 1.3 29.1 ± 1.4 Follow up 30.1 ± 1.3 29.3 ± 1.0 30.1 ± 1.3 29.3 ± 1.0 MCHC (g/L) Baseline 329 ± 32 342 ± 11 341 ± 8 341 ± 13 Follow up 340 ± 8 323 ± 43 340 ± 8 323 ± 43 Neutrophils (cells 10*9/L) Baseline 4.0 ± 0.9 3.7 ± 1.9 4.2 ± 0.9 4.1 ± 2.2 Follow up 4.2 ± 1.2 3.9 ± 1.7 4.2 ± 1.3 3.9 ± 1.7 Lymphocytes (cells 10*9/L) Baseline 1.8 ± .6 2.2 ± 0.7 1.7 ± 0.7 2.2 ± 0.7 Follow up 2.1 ± .3 2.2 ± .8 2.1 ± 0.3 2.2 ± 0.8 Monocytes (cells 10*9/L) Baseline 0.5 ± 0.1 0.6 ± 0.3 0.5 ± 0.2 0.6 ± 0.3 Follow up 0.5 ± .1 0.6 ± 0.3 0.5 ± 0.1 0.6 ± 0.3 44 Ketone (10, 5) Placebo (10, 7) Ketone (5) Placebo (7) Eosinophils (cells 10*9/L) Baseline 0.2 ± .1 0.2 ± 0.2 0.2 ± 0.1 0.2 ± 0.2 Follow up 0.1 ± 0.0 0.2 ± 0.1 0.1 ± 0.0 0.2 ± 0.1 Basophils (cells 10*9/L) Baseline 0.1 ± 0.0 0.0 ± 0.0 0.1 ± 0.0 0.1 ± 0.0 Follow up 0.1 ± 0.1 0.1 ± 0.1 0.1 ± 0.1 0.1 ± 0.1 CRP (mg/dL) Baseline 4.2 ± 5.5 4.5 ± 6.6 2.5 ± 2.6 5.7 ± 7.7 Follow up 1.7 ± 0.9 4.5 ± 8.0 1.7 ± 0.9 4.5 ± 8.0 Data presented as mean ± standard deviation and (n) sample size per outcome and timepoint. Left columns show results for all data available by condition (n=baseline, n=follow up), right columns show results for paired data at both time points. Table 6. Preliminary effect produced by the consumption of thrice a day for 90 days exogenous ketone supplement compared to a non-caloric taste matched placebo in people living with T2D showing baseline and follow-up readings for anthropometric records as waist circumference. Ketone (9, 6) Placebo (10, 8) Ketone (6) Placebo (8) Waist circumference (cm) Baseline 112 ± 19 105 ± 12 107 ± 20 106 ± 13 Follow up 105 ± 20 100 ± 11 105 ± 20 100 ± 11 Data presented as mean ± standard deviation and (n) sample size per outcome and timepoint. Left columns show results for all data available by condition (n=baseline, n=follow up), right columns show results for paired data at both time points. 45 Table 7. Preliminary effect produced by the consumption of thrice a day for 90 days exogenous ketone supplement compared to a non-caloric taste matched placebo in people living with T2D showing baseline and follow-up readings for cardiovascular indicators as systolic and diastolic blood pressure. Ketone (8, 6) Placebo (9, 8) Ketone (6) Placebo (7) Systolic blood pressure (mmHg) Baseline 123 ± 8 120 ± 9 121 ± 2 120 ± 7 Follow up 124 ± 12 119 ± 10 124 ± 12 118 ± 10 Diastolic blood pressure (mmHg) Baseline 79 ± 8 80 ± 7 77 ± 6 80 ± 6 Follow up 77 ± 11 78 ± 5 77 ± 11 77 ± 5 Data presented as mean ± standard deviation and (n) sample size per outcome and timepoint. Left columns show results for all data available by condition (n=baseline, n=follow up), right columns show results for paired data at both time points. Table 8. Mean scores for theory of planned behavior visual analog scale (VAS) survey related to the timepoint and condition of the intervention. Ketone (10, 6) Placebo (10, 8) Ketone (6) Placebo (8) Mean score Baseline 96 ± 7 83 ± 30 95 ± 8 91 ± 6 Follow up 63 ± 34 64 ± 36 63 ± 34 64 ± 36 Data presented as mean ± standard deviation. Scores ranging from 0-100 being 0 lowest likelihood to comply with intervention and 100 greatest likelihood to comply with intervention. Left columns show results for all data available by condition (n=baseline, n=follow up), right columns show results for paired data at both time points. Table 9. Mean scores for Godin 7-day physical activity survey related to the timepoint and condition of the intervention. Ketone (10, 6) Placebo (10, 8) Ketone (6) Placebo (8) Mean score Baseline 1.8 ± 0.8 1.9 ± 0.6 2.0 ± 0.9 1.9 ± 0.6 Follow up 2.3 ± 0.8 1.9 ± 0.8 2.3 ± 0.8 1.9 ± 0.8 Data presented as mean ± standard deviation. Scores ranging from 1-3, with a score of 1 meaning “no/rare” physical activity and 3 being “plenty” physical activity for the last 7 days. Left columns show results for all data available by condition (n=baseline, n=follow up), right columns show results for paired data at both time points. 46 Table 10. Mean scores for hunger visual analog scale (VAS) survey related to the timepoint and condition of the intervention. Ketone (10, 6) Placebo (10, 8) Ketone (6) Placebo (8) VAS mean score Baseline 57 ± 20 59 ± 13 37 ± 10 45 ± 13 Follow up 45 ± 18 44 ± 18 45 ± 14 60 ± 14 Data presented as mean ± standard deviation. Scores ranging from 0-100 with scores closer to 0 meaning the least amount of hunger and scores closer to 100 the greatest level of hunger. Left columns show results for all data available by condition (n=baseline, n=follow up), right columns show results for paired data at both time points. Table 11. Mean scores for fullness visual analog scale (VAS) survey related to the timepoint and condition of the intervention. Ketone (10, 6) Placebo (10, 8) Ketone (6) Placebo (8) VAS mean score Baseline 54 ± 24 59 ± 14 36 ± 13 43 ± 13 Follow up 45 ± 18 44 ± 18 46 ± 15 60 ± 14 Data presented as mean ± standard deviation. Scores ranging from 0-100 with scores closer to 0 meaning the least level of satiety and scores closer to 100 the greatest level of satiety. Left columns show results for all data available by condition (n=baseline, n=follow up), right columns show results for paired data at both time points. Table 12. Mean scores for food cravings questionnaire related to the timepoint and condition of the intervention. Ketone (10, 6) Placebo (10, 8) Ketone (6) Placebo (8) Mean score Baseline 55 ± 10 56 ± 10 56 ± 8 56 ± 9 Follow up 54 ± 14 49 ± 8 54 ± 14 49 ± 8 Data presented as mean ± standard deviation. Scores ranging from 0-100 with scores closer to 0 meaning the least and score closer to 100 meaning the most urges to eat outside of planned meals (cravings). Left columns show results for all data available by condition (n=baseline, n=follow up), right columns show results for paired data at both time points. 47 Table 13. Mean scores for EQ-5D-5L VAS survey related to the timepoint and condition of the intervention. Ketone (10, 6) Placebo (10, 8) Ketone (6) Placebo (8) VAS mean score Baseline 66 ± 20 71 ± 13 69 ± 18 69 ± 14 Follow up 74 ± 12 69 ± 19 74 ± 12 69 ± 19 Data presented as mean ± standard deviation. Scores ranging from 0-100 with scores closer to 0 meaning worst self-perception of general health from participant, scores closer to 100 meaning best self-perception of general health from participant. Left columns show results for all data available by condition (n=baseline, n=follow up), right columns show results for paired data at both time points. Table 14. Mean scores for drink acceptability survey related to the timepoint and condition of the intervention. Ketone (8, 6) Placebo (8, 8) Ketone (6) Placebo (6) Likert mean score Baseline 4.7 ± 2.1 4.6 ± 2.0 5.4 ± 1.0 4.7 ± 2.0 Follow up 3.9 ± 2.6 4.4 ± 2.5 3.9 ± 2.6 5.0 ± 2.2 Data presented as mean ± standard deviation. Scores ranging from 0-7 in a Likert scale, with scores closer to 0 meaning worst acceptability of the supplement and scores closer to 7 the most acceptability of the supplement. Left columns show results for all data available by condition (n=baseline, n=follow up), right columns show results for paired data at both time points. Table 15. Mean scores for gastrointestinal distress survey related to the timepoint and condition of the intervention. Ketone (9, 6) Placebo (8, 8) Ketone (6) Placebo (6) VAS mean score Baseline 18 ± 26 22 ± 17 5 ± 6 20 ± 17 Follow up 19 ± 17 17 ± 13 19 ± 17 18 ± 15 Data presented as mean ± standard deviation. Scores ranging from 0-100 in a VAS with scores closer to 0 meaning no GI distress symptoms related to supplement consumption and scores closer to 100 the most GI distress symptoms related to supplement consumption. Left columns show results for all data available by condition (n=baseline, n=follow up), right columns show results for paired data at both time points. 48 Table 16. Results for ASA24 dietary assessment related to timepoint and condition of the intervention. Ketone (9, 6) Placebo (10, 8) Ketone (6) Placebo (8) Available data Paired data Total caloric intake (kcal) Baseline 1,763.0 ± 914.5 2,175 ± 769.7 1,311.1 ± 631.0 2,094.3 ± 818.6 Follow up 1,132.6 ± 667.0 1869.3 ± 874.2 1,132.6 ± 667.0 1,869.3 ± 874.2 Total protein intake (g) Baseline 96.3 ± 44.4 103.3 ± 46.4 72.8 ± 26.8 102.3 ± 52.5 Follow up 61.7 ± 36.0 106.8 ± 63.7 61.7 ± 36.0 106.8 ± 63.7 Total lipid/fat intake (g) Baseline 85.3 ± 50.4 90.7 ± 41.3 57.9 ± 26.3 86.7 ± 35.1 Follow up 53.1 ± 38.5 90.9 ± 44.7 53.1 ± 38.5 90.9 ± 44.7 Total carbohydrate intake (g) Baseline 155.6 ± 84.7 219.8 ± 83.5 130.5 ± 83.8 216.4 ± 94.3 Follow up 106.5 ± 71.1 157.6 ± 90.1 106.5 ± 71.1 157.6 ± 90.1 Data presented as mean ± standard deviation and (n) sample size per outcome and timepoint. Left columns show results for all data available by condition (n=baseline, n=follow up), right columns show results for paired data at both time points. 49 Table 17. Preliminary effect produced by the consumption of thrice a day for 90 days exogenous ketone supplement compared to a non-caloric taste matched placebo in people living with T2D showing baseline (days -5-0), Active 1 (days 1-9) and follow-up (days 77-90) readings for continuous glucose monitor metrics. Ketone (7, 7, 4) Placebo (9, 9, 8) Ketone (4) Placebo (7) Available data Paired data Average glucose (mmol/L) Baseline 9.5 ± 4.3 7.4 ± 1.6 9.5 ± 5.8 7.8 ± 1.6 Active 1 7.9 ± 2.4 7.2 ± 1.3 7.9 ± 3.1 7.5 ± 1.3 Active 2 6.8 ± 1.8 7.4 ± 1.3 6.7 ± 1.8 7.6 ± 1.3 Estimated Hb A1c (%) Baseline 7.6 ± 2.7 6.3 ± 1.0 7.6 ± 3.7 6.5 ± 1.0 Active 1 6.6 ± 1.5 6.2 ± 0.8 6.6 ± 1.9 6.3 ± 0.8 Active 2 5.9 ± 1.1 6.3 ± 0.8 5.9 ± 1.1 6.4 ± 0.8 AUC (mmol h/L) Baseline 7.0 ± 3.1 5.5 ± 1.2 6.9 ± 4.2 5.7 ± 1.2 Active 1 5.8 ± 1.8 5.3 ± 0.9 5.9 ± 2.3 5.5 ± 1.0 Active 2 4.9 ± 1.2 5.5 ± 1.0 4.9 ± 1.2 5.7 ± 1.0 LBGI (mmol/L) Baseline 0.4 ± 0.7 0.4 ± 0.5 0.7 ± 1.0 0.4 ± 0.6 Active 1 0.5 ± 0.7 0.3 ± 0.3 0.7 ± 0.8 0.3 ± 0.3 Active 2 0.8 ± 0.7 0.3 ± 0.4 0.8 ± 0.7 0.2 ± 0.1 HBGI (mmol/L) Baseline 9.9 ± 14.0 2.8 ± 3.3 11.2 ± 19.1 3.5 ± 3.5 Active 1 4.6 ± 5.3 2.1 ± 2.4 5.5 ± 6.8 2.6 ± 2.5 Active 2 2.0 ± 2.8 2.6 ± 2.9 2.0 ± 2.8 2.8 ± 3.0 MAGE Baseline 3.9 ± 1.5 3.7 ± 1.4 3.5 ± 1.8 4.2 ± 1.2 Active 1 3.8 ± 2.0 3.2 ± 1.0 3.8 ± 2.6 3.5 ± 1.0 Active 2 3.1 ± 1.5 3.9 ± 1.1 3.1 ± 1.5 4.0 ± 1.2 Data presented as mean ± standard deviation and (n) sample size per outcome and timepoint. Left columns show results for all data available by condition (n=baseline, n=active 1, n=active 2), right columns show results for paired data at both time points. 50 Discussion Overview The study was a 90-day parallel-arm remotely conducted randomized, double-blind pilot trial comparing thrice daily consumption of an exogenous ketone supplement and a taste-matched placebo in people living with T2D. Overall, recruitment goals were achieved and the study was conducted as planned. Mixed results on feasibility were found as there was a high rate of drop-out (40%, or 4 of 10 participants) in the exogenous ketone supplement group, largely due to gastrointestinal issues, yet participants who remained in the trial had high compliance. Despite the higher-than-expected drop-out, data availability at the follow-up timepoint for those that remained in the trial was generally high (80-93%) suggesting that the remote trial procedures are feasible. Recruitment methods, rate, and randomization Aiming to fulfill recruitment goals for this trial we focused on three main tools: reaching out to participants from previous trials that our laboratory had conducted, posting advertisements online on local sites and newspapers, and hiring a third-party company (WayTurn) in charge of attracting potential participants by means of social media advertising, filtering, and sending them our way for screening. Perhaps not surprisingly, out of the 38 participants that were screened for eligibility, 28 of them (74%) were referred by the hired company. From the total participants that ended up enrolling in the trial, 85% (17) were from the WayTurn referrals. Although not captured or explicitly quantified in this pilot trial, the cost per enrolled participant would be an important consideration for planning future trials using this method of recruitment. WayTurn works by assigning an individual rate per referred participant that is determined according to the difficulty of and narrowness of inclusion criteria, exclusion criteria, demographics, location, length of the trial, 51 type of intervention, and other metrics not fully disclosed to the client. For this trial, WayTurn delivered a quotation for $142.43 CAD per referral. The company successfully delivered 33 referrals; however, just over 50% of them were successfully enrolled after further screening. Reasons for failure to enroll varied from not fitting the inclusion and/or exclusion criteria upon a better screening with the researcher to not picking up the phone. Considering this conversion rate, the total cost for each enrolled participant with this company was $276.48. Although success in enrolling referrals can vary widely and would be dependent on the full context (e.g., study design, location, timing), taking this cost into consideration may be important for planning of a potential future efficacy trial of exogenous ketone supplements in T2D. The successful recruitment rate for this trial was set at a completion timeline of 5 months or less, estimating a monthly enrollment of 4 participants per month. With the first participant being enrolled on June 29th, 2023, and the last participant coming into the trial December 11th, 2023, our recruitment goal fell short by 12 days. However, we are confident that if we fully depended on the third-party recruitment company, recruitment could have proceeded at a much faster rate. Request for referrals from WayTurn was done at researchers’ discretion, trying to stretch and optimize funding, and not request more referrals than strictly necessary. We presume that in a larger trial, where a higher number of participants would be needed and likely multiple sites would be included, handling excess referrals by the research team could be accounted for and the enrollment process could be done at a faster pace. However, close monitoring of incoming referrals (which cost money) with successful contact and conversion to enrolled participants is important for researchers to consider when using third party recruiting services. Closing this topic, it is important to note that the conversion rate was much lower in screened participants that did not come from the third-party company, as they did not undergo a previous screening. The enrollment rate for these leads was 30% (3 out of 10), and whilst WayTurn sent over the referrals at two well delimited timepoints of about 52 30 days in total, researchers were in active search of participants using the previously mentioned tools for the whole duration of recruitment period (5.5 months). This makes it evident that without hiring a successful third-party recruitment service, it would have been virtually impossible to accomplish recruitment if using the more passive approaches of past participants, poster advertisements, and word-of-mouth. Compliance, feasibility, and acceptability Participants were prompted weekly with a single question survey regarding the number of drinks/supplements they had consumed during the last 7 days. Successful compliance rate for this trial was set at 67% (i.e., ~2 out of 3 drinks per day). It is important to mention that adequate compliance rate varies between trials, product type, population, and living condition (inside or outside hospital/laboratory facilities). Studies addressing compliance will typically analyze these multiple factors and set a value that is presumably logical and significant for their design. In general, adequate compliance will be set somewhere between 60-80% of prescription achievement, with values higher than 80% considered strong compliance and values lower than 60% considered inadequate/fail to comply (51) (52). Our 67% compliance rate was set considering that participants would be asked to consume the supplement thrice per day, allowing them to miss one drink per day. Our results show that participants, on average, were consistently above this compliance rate with the lowest percentage being at the end of the trial for the placebo group with 67%. As shown in figure 6, average compliance amongst both groups was always above the 67% threshold. Considering figure 6 we can also notice that after an initial decrease in compliance for the first 5 weeks for the experimental group, consumption rate stabilizes close to 15 drinks per week (~70%). Although with some more variability, we can also see this stabilization effect after week 5 for the control group, with compliance rates close to 85%. It is not surprising to see somewhat higher 53 compliance at the start of the trial with more realistic compliance rates stabilizing between weeks 5-12. Overall, this relatively high level of compliance maintained across the 90-day trial is encouraging and suggests that longer-term consumption of the concentrated ketone drink might be feasible. Assessment tools completion rates Related to implemented assessment tools, previous literature has reported completion rates ranging from 60-80%, with this higher percentage being usually considered good practice for pharmaceutical studies (53). As shown in Table 3, each of our outcome variables being surveys, blood work, anthropometric and self-reported blood pressure had a high compliance ranging from 80-100% of actively enrolled participants. Thus, the various different assessment tools used in this study appeared to be efficient and acceptable for participants to complete. However, related to the free-living environment of core of this study, there are aspects that we could encourage to do differently for future bigger trials. • Surveys and questionnaires: Evidence shows that e-mail response rates have decreased in the past decades, and successful response rate for e-mail sent surveys without follow-up prompts can be as low as 25-30% (53). However, e-mail sent surveys used alongside other communication tools such as text messages or phone calls can increase response rates to 70% (53). Although our formal communication pathway with participants was merely via e-mail, the small sample size made it easy for researchers and participants to be aware of individual situations and participants were encouraged to contact the researchers directly upon any issues. Nevertheless, participants in a bigger trial will likely be located more remotely and, thus, a smaller engagement can be expected which will require to develop strategies to avoid loss of information or low response rates. 54 • Supplement compliance: Response rate to weekly compliance was consistently above 80% over the 13 weeks of trial. However, clinical trials will usually confirm this information requesting empty pill blisters or, in this case, empty supplement bottles (54). Since the main outcome of this trial was not intervention efficacy, we decided to not retrieve empty bottles from participants to simplify logistics. However, we encourage that further and bigger research should consider implementing a stronger compliance oversight strategy. • Blood work/laboratory analysis: This assessment tool may be complicated to change in future trials since it will vary within geographical areas and/or different health systems. Our baseline efficacy to collect this outcome was 100% since it was the final indication before participants could be formally enrolled and randomized into our trial. However, we did see a lower rate of compliance for the follow-up period with a total of 12 laboratory results sent over to our office out of the 15 expected. Two of these missing results had to do with participant’s personal reasons, which researchers assume could be owed to lack of willingness or ability to obtain a blood draw. Since as per Ethics Research Board indications, researchers are not allowed to link any personal information to the research outcomes, hence, participants did not have access to their laboratory results. Upon personal critical thinking, we believe that having a way to share this information with participants could have a positive impact upon increasing efficacy of data collection. • Continuous glucose monitoring: Two issues occured regarding this assessment tool for this study. Failure to collect 100% of the data for CGM was due to either 1) sensor stopped working after the first (not accurately quantified) days of usage or 2) incompatibility with participants personal cell phone. After further conversations with our colleagues both issues appear not to be common; the sensor chosen or this trial was selected upon good reliability on previous trial conducted by our team. However, a slight improvement that could be made 55 is to implement a strategy for sensor failure and prompt reaction and potentially consider confirming phone compatibility prior to enrolment. Preliminary efficacy As a pilot feasibility study, we were not powered to provide reliable statistical analysis and report results’ significance. Regarding withdrawal rate, we were unable to run simple statistics or calculate effect size. Thus, results are to be interpreted with caution. However, they do show interesting trends related to blood glucose control outcomes assessed using HbA1c and CGM. Within our results, we noticed a mean decrease of ~ ~1.2% (using paired data) in HbA1c within the ketone group when comparing baseline to follow-up timepoints. In the context of T2D, pharmacological monotherapies currently aims for a reduction of 0.5-1.5% in HbA1c levels (55). Many current treatment paradigms aim for achieving HbA1c levels of less than 7.0% (55). Adding to this, a recent meta-analysis has shown that a decrease of 0.5% in HbA1c levels is significantly associated with a decrease in major adverse cardiac events (MACE) (56). Given this, if the tendency for a reduction in HbA1c seen in this pilot trial could be extrapolated to a larger sample size within an efficacy trial this could firmly demonstrate ketone supplementation as a bona fide T2D treatment. However, it is important to not overstate the findings seen given the small sample size and high drop-out rate in the ketone group. A larger, properly powered future RCT would be needed to provide sufficient information that can lead to stronger arguments upon the efficacy of exogenous ketone supplementation in the context of T2D. Strengths, limitations, and future directions This trial has shown mixed results, which fulfill the core intention of running a pilot feasibility study. On the one hand, longer-term compliance to consume an exogenous ketone supplement, previously shown to significantly increase blood ketones and lower glucose, showed strong signs of 56 being feasible in those participants who remained in the trial. However, the high withdrawal rate and the apparent difference for this outcome within study groups should be noted. Even though our placebo was attempted to be matched for taste, appearance, and pH, it probably did not match the exact taste and level of gastrointestinal discomfort caused by the nature of ketone bodies. Nevertheless, compliance as shown in Figure 6 is promising. Three out of four participants that withdrew from the ketone group did so within the first 7 days of supplementation, one of them did so on day 1 and another on day 4. We interpret this as “high” responders to gastrointestinal distress symptoms that ketone supplements may cause. However, the already mentioned stabilization effect seen after week 3-5, could help with potential modification in a future study design to ensure better participant retention (e.g., characterizing a participant as intolerant to ketone supplementation upon an initial tolerability/run-in phase). Addressing this factor, to date, many commercial brands are seeking further improvements in the flavor and drinkability of this supplement. However, no company and/or research team has been able to create a highly palatable product after >12 years since the first human safety and tolerability study (23). This suggests the possibility of not advancing much further and potentially accepting that exogenous ketone supplementation will always have a harsh taste and polarized acceptability. It is important to note that the mechanism of action responsible for potential blood glucose lowering effect is not clear yet. An important line of thinking potentially links ketone supplementation slowing gastric emptying and/or delaying glucose absorption or potentially having an impact in appetite control, thus decreasing postprandial glucose excursions (19) (41). However, this trial did not strictly control for consumption of the supplement related to food intake. Although participants were asked and encouraged to consume the supplement before every big meal (breakfast, lunch, and dinner) this was not mandatory and given the free-living nature of the trial was not captured. Our exploratory dietary intake assessment was conducted using the 24-hour self-57 reported dietary recall, which is likely not sensitive enough to detect any (subtle) changes in dietary intake that may have resulted from consuming the ketone (or placebo) supplement. Although based on self-report, there were no apparent trends for differences between groups in waist circumference, which suggests that energy intake or balance were likely not impacted by the supplement regimen in this trial. However, future studies may want to consider more detailed analyses of how ketones impact appetite, energy intake, and energy balance. Ketone supplementation keeps befuddling researchers, and this trial was no exception. Logistic implementation of the study was a big challenge, but we are confident that our findings and suggestions for improvements can set the basis for bigger and better trials. The trends that we found for blood glucose control are encouraging and justify for deeper analysis inside and outside laboratory facilities for exogenous ketone supplementation in the context of T2D. The same way that we decided to take a step back from more mechanistic laboratory research, we believe that for a further bigger trial, there would be a need to target a specific mechanism of action, with a potential dose-response study, that would help us not only replicate the glucose-lowering effect but to determine how exogenous ketones might be working to lower glucose. We have successfully conducted a pilot trial that gives an important test of logistics and key aspects of conducting a remote prolonged free living environment study design. Combining this knowledge with new understandings on how exogenous ketone bodies influence glycemic regulation will set the tone for future development of a clinical trial with efficacy as its primary outcome. 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Am J Clin Nutr. 2019; 110: 1491-1501. 64. Monteyne AJ, Falkenhain K, Whelehan G, Neudorf H, Abdelrahman DR, Murton AJ, Wall BT, Stephens FB, Little JP. A ketone monoester drink reduces postprandial blood glucose concentrations in adults with type 2 diabetes: a randomised controlled trial. Diabetologia. 2024; 67(6): 1107-1113. 64 Appendices Appendix A. Health screening questionnaire Health Screening Questionnaire Please complete the survey below. Thank you! Page 1 1) Have you been diagnosed with any of the following Heart Attack (past or current)? Check all that apply Heart/Blood Vessel Condition Stroke High Blood Pressure Diabetes Hormone or Thyroid Condition Kidney Disease Cognitive Impairment/Memory Condition 2) How long have you been diagnosed with type 2 diabetes? 3) Please list any medications or vitamin/mineral supplements you are currently taking, including dosages: 4) Sex: Male Female Other 5) Month and Year of Birth: 6) Age: 7) Self-reported weight (please indicate kg or lbs): 8) Self-reported waist circumference (indicate cm or in): 9) Self-reported height (please indicate m/cm or ft/in): 10) What is your average blood pressure? 2022-08-03 15:33:47 projectredcap.org 65 Appendix B. Theory of planned behavior questionnaire Confidential Page 1 2022-04-27 11:53:32 projectredcap.org For me, consuming a ketone drink 3 times per day over the next 3 months would be... TPB Questionnaire Please complete the survey below. Thank you! Please try to answer the following questions as honestly as possible. For each statement, please select a number from 1 to 7. 1 strongly disagree 2 3 4 5 6 7 strongly agree 1) 1 strongly disagree 2 3 4 5 6 7 strongly agree 2) 1 useless 2 3 4 5 6 7 useful 3) 1 unimportan t 4) 2 3 4 5 6 7 important 1 worthless 2 3 4 5 6 7 valuable 5) 1 not worthwhile 6) 2 3 4 5 6 7 worthwhile 1 harmful 2 3 4 5 6 7 beneficial 7) For me, consuming a ketone drink 3 times per day over the next 3 months would be... For me, consuming a ketone drink 3 times per day over the next 3 months would be... For me, consuming a ketone drink 3 times per day over the next 3 months would be... For me, consuming a ketone drink 3 times per day over the next 3 months would be... Most people in my social network regularly consume a ketone drink: People who are important to me would approve of me consuming a ketone drink 3 times per day over the next 12 weeks: 66 For me, consuming a ketone drink 3 times per day over the next 3 months would be... For me, consuming a ketone drink 3 times per day over the next 3 months is extremely: 1 unsatisfyin g 8) 2 3 4 5 6 7 satisfying 1 unpleasant 9) 2 3 4 5 6 7 pleasant 10) 1 not enjoyable 2 3 4 5 6 7 enjoyable 1 boring 2 3 4 5 6 7 exciting 11) 1 not fun 2 3 4 5 6 7 fun 12) 1 difficult 2 3 4 5 6 7 easy 13) 1 strongly disagree 2 3 4 5 6 7 strongly agree 14) 1 strongly disagree 2 3 4 5 6 7 strongly agree 15) If I want to, I could easily consume a ketone drink 3 times per day over the next 3 months. I believe that I have control over whether I do or do not consume a ketone drink 3 times per day over the next 3 months. For me, consuming a ketone drink 3 times per day over the next 3 months would be... For me, consuming a ketone drink 3 times per day over the next 3 months would be... For me, consuming a ketone drink 3 times per day over the next 3 months would be... For me, consuming a ketone drink 3 times per day over the next 3 months would be... 67 1 not at all 2 3 4 5 6 7 very regularly 16) 1 definitely false 2 3 4 5 6 7 definitely true 17) The following questions pertain to consuming a ketone drink consistently 3 times per day. Try to answer as honestly as possible and please select a value from 0 to 100% for this question. Over the next 3 months, how confident are you that you can consume a ketone drink 3 times per day ... I intend to consume a ketone drink 3 times per day over the next 3 months. I intend to consume a ketone drink with the following regularity per week over the next 3 months: 68 Appendix C. Drink acceptability questionnaire Description: CIHR 3-month Ketone Trial; Study Number: H22-00644 Version 1: 9 June 2022 1 Drink Acceptability Questionnaire Please complete the survey below. Thank you! 4) How much effort does it take for you to consistently consume this drink? A lot of effort Neutral No Effort 5) How satisfied or dissatisfied do you feel after consuming this drink? Very dissatisfied Neutral Very satisfied 6) Overall, how satisfied or dissatisfied are you with consuming this drink? Very dissatisfied Neutral Very satisfied 7) How easy or difficult would it be for you to continue with consuming this drink? Very Difficult Neutral Very Easy (Place a mark on the scale above) 69 Appendix D. Godin physical activity questionnaire Description: CIHR 3-month Ketone Trial; Study Number: H22-00644 Version 1: 9 June 2022 1 GODIN Physical Activity Please complete the survey below. Thank you! During a typical 7-day period (a week), how many times on the average do you do the following kinds of exercise for more than 15 minutes during your free time (write on each line the appropriate number). 1) STRENUOUS EXERCISE (HEART BEATS RAPIDLY) __________________________________ (e.g., running, jogging, hockey, football, soccer, squash, basketball, cross-country skiing, judo, roller skating, vigorous swimming, vigorous long-distance bicycling) 2) 3) 4) During a typical 7-day period (a week), in your leisure time, how often do you engage in any activity long enough to work up a sweat (heart beats rapidly)? OFTEN SOMETIMES RARELY/NEVER MODERATE EXERCISE (NOT EXHAUSTING) (e.g., fast walking, baseball, tennis, easy bicycling, volleyball, badminton, easy swimming, alpine skiing, popular and folk dancing) __________________________________ MILD EXERCISE (MINIMAL EFFORT) (e.g., yoga, archery, fishing from river bank, bowling, horseshoes, golf, snow-mobiling, easy walking) __________________________________ 70 Appendix E. EQ-5D-5L questionnaire EQ-5D-5L Please complete the survey below. Thank you! Page 1 The following questions ask about your general health. Please indicate the statements that best describe your own state of health today. Check only one box for each category. 1) Mobility I have no problems in walking about I have slight problems in walking about I have moderate problems in walking about I have severe problems in walking about I am unable to walk about 2) Self-care I have no problems washing or dressing myself I have slight problems washing or dressing myself I have moderate problems washing or dressing myself I have severe problems washing or dressing myself I am unable to wash or dress myself 3) Usual activities I have no problems doing my usual activities I have slight problems doing my usual activities I have moderate problems doing my usual activities I have severe problems doing my usual activities I am unable to do my usual activities 4) Pain/discomfort I have no pain or discomfort I have slight pain or discomfort I have moderate pain or discomfort I have severe pain or discomfort I have extreme pain or discomfort 5) Anxiety/Depression I am not anxious or depressed I am slightly anxious or depressed I am moderately anxious or depressed I am severely anxious or depressed I am extremely anxious or depressed 6) To help people say how good or bad their state of health is, we have drawn a scale (rather like a thermometer) on which the best state you can imagine is marked 100 and the worst state you can imagine is marked 0. We would like you to indicate on this scale how good or bad your own health is today, in your opinion. Please do this by drawing a line from the box below to whichever point on the scale indicates how good or bad your state of health is today. Your state of health today: 0 100 (Place a mark on the scale above) 2022-08-03 15:34:30 projectredcap.org 71 Appendix F. Food cravings questionnaire. Cravings Please complete the survey below. Thank you! Page 1 1) How often do you experience strong urges to eat particular types of food? Never All the time (Place a mark on the scale above) 2) On average how often do you experience a strong urge Several times a to eat a particular type of food? day Once a month (Place a mark on the scale above) 3) How strong are these urges you experience to eat particular types of food? Extremely weak Extremely strong (Place a mark on the scale above) 4) Are the experiences of strong urges to eat a particular food always of the same strength? Never Always (Place a mark on the scale above) 5) How easy is it to ignore this strong urge to eat a particular food? Very easy Impossible (Place a mark on the scale above) 6) Is a strong urge to eat a particular food the same as a craving for food? No Yes (Place a mark on the scale above) 7) If not, please describe in what way it is different: 2022-08-03 15:34:28 projectredcap.org 72 Appendix G. VAS Hunger and Fullness VAS Hunger and Fullness Please drag the button horizontally to indicate your response to each question. Thank you! Page 1 1) How hungry do you feel? I am not hungry I have never been at all hungrier (Place a mark on the scale above) 2) How satisfied do you feel? I am completely I cannot eat empty another bite (Place a mark on the scale above) 3) How full do you feel? Not at all full Totally full (Place a mark on the scale above) 4) How much food do you think you can eat? Nothing at all A lot (Place a mark on the scale above) 5) Would you like to eat something sweet? No, nothing at Yes, very much all (Place a mark on the scale above) 6) Would you like to eat something salty? No, nothing at Yes, very much all (Place a mark on the scale above) 7) Would you like to eat something savoury? No, nothing at Yes, very much all (Place a mark on the scale above) 8) Would you like to eat something fatty? No, nothing at Yes, very much all (Place a mark on the scale above) 2022-08-03 15:32:50 projectredcap.org 73 Appendix H. Overall acceptability questionnaire Document: Overall acceptability questionnaire A. How would you describe your overall experience participating in this trial? B. Related to the supplement, what is your perceived opinion in terms of drinkability? C. If the supplement was recommended by your physician because of its positive effects related to blood sugar control, would you agree to consume it daily? D. Is there anything that could make it easier to use this as a regular consumption supplement? E. Please help us with any other opinion and/or feedback that you would like us to know.
Thesis/Dissertation
eng
Health and Exercise Sciences
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Little, Jonathan P.
Examining the feasibility of prolonged ketone supplement drink consumption in adults living with type 2 diabetes
Text