The Effect of Different Ketone Supplements on Blood Beta-hydroxybutyrate (β-OHB) and Blood Glucose in Healthy Individuals
Phase: NA
Conditions: Ketosis, Blood Glucose
Sponsor: University of British Columbia

 
 
 
The Effect of Different Ketone Supplements on Blood β-OHB and Blood 
Glucose in Healthy Individuals 
 
Version 2 – Feb 15, 2022 [H22-00083] – NCT05273411 
 
1
Faculty of Health and Social Development 
School of Health and Exercise Sciences 
3333 University Way 
Kelowna, BC V1V 1W5 
 
Research Question 
 
How do different ketone supplement types affect blood beta-hydroxybutyrate 
(β-OHB) and blood glucose in healthy individuals? 
 
Objectives 
a) To determine the effect of acute ketone supplementation on blood β-
OHB across different types of ketone supplements. 
b) To compare changes in blood glucose following acute supplementation 
with different types of ketone supplements. 
c) To evaluate the influence of acute ketone supplementation on blood 
pressure and heart rate across different types of ketone supplements. 
d) To investigate the effect of acute supplementation with different ketone 
supplements on gastrointestinal discomfort, hunger, and fullness. 
e) To explore supplement acceptability and taste across different ketone 
supplements. 
 
Hypotheses 
a) All ketone supplements will raise blood β-OHB. 
b) All ketone supplements will lower blood glucose. 
This is an exploratory/descriptive pilot study that is designed to inform future 
research; therefore, we do not pose specific hypotheses as to how the ketone 
supplements will differ with respect to their effects on the above-outlined 
objectives. 
 

 
 
 
The Effect of Different Ketone Supplements on Blood β-OHB and Blood 
Glucose in Healthy Individuals 
 
Version 2 – Feb 15, 2022 [H22-00083] – NCT05273411 
 
2
Faculty of Health and Social Development 
School of Health and Exercise Sciences 
3333 University Way 
Kelowna, BC V1V 1W5 
 
Background Information 
Acute increases in blood ketones (beta-hydroxybutyrate, β-OHB) have been found 
to temporarily reduce blood glucose [1] but therapeutic application has traditionally 
been limited due to the need for ketone infusion or severe dietary restrictions to 
achieve nutritional ketosis. With the recent development of exogenous ketone 
supplements that can be consumed orally [2], it is now possible to acutely raise 
blood β-OHB concentration without the need for other nutritional modifications or 
invasive procedures. In our lab, we have previously shown that acute ingestion of 
oral ketone supplements potently increases β-OHB and immediately lowers blood 
glucose [3-6]. These findings suggest exogenous ketones to be a promising 
therapeutic strategy aimed at lowering blood glucose and improving blood glucose 
control in individuals with impaired glucose metabolism, particularly individuals 
with prediabetes or type 2 diabetes [7].  
The first ketone supplements available on the market were in the form of either 
ketone salts (i.e., β-OHB bound to a mineral salt) or ketone monoesters (i.e., β-
OHB bound to a ketone precursor such as butanediol) [2]. Ketone salts can lead 
to gastrointestinal discomfort in many individuals [8] and ketone monoesters have 
an extremely unpleasant bitter taste. This makes adherence to a nutritional 
supplement regimen using ketone salts or monoesters challenging. Since then, 
new formulations have been developed that differ with respect to ingredients (e.g., 
include flavouring to mask the bitter taste and improve palatability) and chemical 
properties (e.g., different precursors and/or types of molecule the β-OHB is bound 
to). In particular, two new ketone supplements (beta-hydroxybutyric acid and 1,3-
Butanediol) have recently become commercially available that promise an 
improved consumer experience.   

 
 
 
The Effect of Different Ketone Supplements on Blood β-OHB and Blood 
Glucose in Healthy Individuals 
 
Version 2 – Feb 15, 2022 [H22-00083] – NCT05273411 
 
3
Faculty of Health and Social Development 
School of Health and Exercise Sciences 
3333 University Way 
Kelowna, BC V1V 1W5 
However, little is known about the pharmacokinetics of these supplements, which 
can be expected to differ from the original supplements based on differences in 
formulation. This, in turn, may affect the glucose-lowering properties of the 
exogenous ketone supplements. While qualities such as an improved taste and 
gastrointestinal tolerability are desirable in ketone supplements, it is of importance 
for the field of research focusing on the therapeutic value of exogenous ketones to 
establish the effect of these different ketone supplements on blood β-OHB and 
blood glucose and determine how they compare to the effects of the by now well-
established ketone monoester.   
The overall aim of this pilot study is therefore to test the β-OHB-raising and 
glucose-lowering effect of these two new types of commercially-available oral 
ketone supplements (beta-hydroxybutyric acid and 1,3-Butanediol) in comparison 
to a ketone monoester, which we have used in our previous studies on ketone 
supplementation. 
 
 
 

 
 
 
The Effect of Different Ketone Supplements on Blood β-OHB and Blood 
Glucose in Healthy Individuals 
 
Version 2 – Feb 15, 2022 [H22-00083] – NCT05273411 
 
4
Faculty of Health and Social Development 
School of Health and Exercise Sciences 
3333 University Way 
Kelowna, BC V1V 1W5 
Research Method 
Experimental design 
A double-blind randomized cross-over pilot study in which N = 12 healthy adults 
will participate in three experimental trials with a different ketone supplement in 
each trial. Each ketone supplement has FDA GRAS (Generally Recognized As 
Safe) status as a food additive and is available for online purchase in Canada: 
1) “KE4” (by KetoneAid) 
• Beta-hydroxybutyrate monoester 
2) “Kenetik” (by DrinkKenetik) 
• Beta-hydroxybutyric acid 
3) “Ketone 2.0” (by HVMN) 
• 1,3-Butanediol  
All participants will provide written informed consent before start of data collection. 
Following screening and informed consent, eligible participants will participate in 
three experimental trials of β-OHB supplementation with the order of treatment 
randomized, balanced, and separated by at least 48 hours. Pre-menopausal 
women will be tested in the follicular phase (days 3-9) to minimize any potential 
changes in insulin sensitivity across the menstrual cycle [9]. Participants will be 
asked to refrain from structured exercise and alcohol on the day before each trial. 
Furthermore, participants will be instructed to record their dietary intake on the day 
prior to their first experimental visit, and to consume the same foods and quantities 
on the days before the subsequent experimental visits. Participants will then be 
asked to fast overnight (at least 8 hours) and refrain from taking any medications 
on the morning of the trial.  
Upon arrival, a baseline fasting blood sample will be obtained via finger prick. The 
oral ketone supplement will then be consumed within 5 minutes. The different 

 
 
 
The Effect of Different Ketone Supplements on Blood β-OHB and Blood 
Glucose in Healthy Individuals 
 
Version 2 – Feb 15, 2022 [H22-00083] – NCT05273411 
 
5
Faculty of Health and Social Development 
School of Health and Exercise Sciences 
3333 University Way 
Kelowna, BC V1V 1W5 
supplement types will have an overall similar yet likely distinguishable flavour and 
will be provided at different volumes (based on the manufacturer’s formulation) – 
however, participants will not be aware which flavour and volume will correspond 
to which ketone supplement. Furthermore, all supplements will be provided in 
opaque plastic bottles (labelled with “A”, “B”, or “C” according to a code that only 
the principal investigator has access to) such that neither the participant nor the 
research lead will be able to visually identify the supplement type. Each ketone 
supplement will be provided at a dose of 10 g active ingredient (i.e., 10 g of beta-
hydroxybutyrate monoester, beta-hydroxybutyric acid, or 1,3-Butanediol). This is 
the recommended serving size for the two new ketone supplements to be tested 
(beta-hydroxybutyric acid and 1,3-Butanediol) and is in line with a previous 
pharmacokinetic study that showed a dose of ~ 10 g beta-hydroxybutyrate 
monoester to successfully raise β-OHB within 30 minutes [2]. Specifically, this 
leads to one trial each with: 
1) 20 mL of “KE4” (i.e.,10 g beta-hydroxybutyrate monoester) 
2) 237 mL of “Kenetik” (i.e., 10 g beta-hydroxybutyric acid) 
3) 35 mL of “Ketone 2.0” (i.e., 10 g 1,3-Butanediol) 
The primary outcome will be total β-OHB as represented by the area under the 
curve across 4 hours. Secondary outcomes include blood glucose, blood pressure, 
heart rate, taste and supplement acceptability, hunger and fullness, and 
gastrointestinal tolerability - all measured across the 240-minute post-
supplementation period. Blood samples will be collected via finger prick; blood β-
OHB and glucose levels will be measured using the “Keto-Mojo” handheld monitor 
at baseline and 15, 30, 60, 90, 120, 180, and 240 minutes after ingestion of the 
ketone supplement. 
 

 
 
 
The Effect of Different Ketone Supplements on Blood β-OHB and Blood 
Glucose in Healthy Individuals 
 
Version 2 – Feb 15, 2022 [H22-00083] – NCT05273411 
 
6
Faculty of Health and Social Development 
School of Health and Exercise Sciences 
3333 University Way 
Kelowna, BC V1V 1W5 
Visits Summary 
The study will involve a total of 3 visits to the laboratory. 
Visit 1: Baseline screening & first experimental trial (duration 270 minutes) 
Individuals will be recruited by word-of-mouth. Individuals must be able to read and 
understand English in order to understand the study instructions. On the initial visit, 
the potential participant will read through the information and consent form, be able 
to ask any questions, and provide written informed consent. The research lead 
(Kaja Falkenhain, PhD Candidate) will ensure that the participant understands the 
details of the study and consent form.  
Inclusion criteria will be: i) over the age of 18; and ii) able to fast overnight. 
Exclusion criteria will include: i) being a competitive endurance athlete; ii) 
following a ketogenic diet, low-calorie diet, periodic fasting regimen, or regularly 
consuming ketogenic supplements; iii) being unable to travel to and from the 
university; iv) being unable to follow the diet instructions; v) being pregnant or 
planning to become pregnant during the study; vi) having been diagnosed with a 
chronic disorder of glucose or fat metabolism, including type 2 diabetes, chronic 
pancreatitis, or gallbladder disease; vii) being unable to read or communicate in 
English.  
Females will complete the menstrual cycle questionnaire to confirm they are in the 
follicular phase. Anthropometric and physiologic measurements will be collected 
(height, body weight, waist circumferences, blood pressure) for baseline 
participant characterization. The research lead will review the dietary record with 
the participant and confirm the participant is fasted and has refrained from 
structured exercise and alcohol the day prior.  
A baseline finger prick will then be performed before ingestion of the ketone 
supplement. Another 7 finger pricks will be performed at 15, 30, 60, 90, 120, 180, 

 
 
 
The Effect of Different Ketone Supplements on Blood β-OHB and Blood 
Glucose in Healthy Individuals 
 
Version 2 – Feb 15, 2022 [H22-00083] – NCT05273411 
 
7
Faculty of Health and Social Development 
School of Health and Exercise Sciences 
3333 University Way 
Kelowna, BC V1V 1W5 
and 240 minutes after ingestion of the supplement. Throughout the 240-minute 
period, participants will be allowed to do quiet work or perform other sedentary 
activities (reading, watching a movie). Participants will be asked to complete the 
questionnaire on gastrointestinal discomfort 60 minutes after ingestion of the 
supplement, and the questionnaires on hunger and fullness as well as taste and 
supplement acceptability at the end of the 240-minute post-supplementation 
period. 
At the end of the visit, the participant will be reminded to consume the same diet 
on the day before the next experimental trial, and to refrain from structured 
exercise and alcohol consumption in the 24 hours prior. 
Visits 2 & 3: Second and third experimental trial (duration 240 minutes) 
The second and third experimental trial will be identical to the first visit (except no 
baseline anthropometric measurements will be performed), with the only difference 
being the different ketone supplement provided to the participant. 
 
 
 

 
 
 
The Effect of Different Ketone Supplements on Blood β-OHB and Blood 
Glucose in Healthy Individuals 
 
Version 2 – Feb 15, 2022 [H22-00083] – NCT05273411 
 
8
Faculty of Health and Social Development 
School of Health and Exercise Sciences 
3333 University Way 
Kelowna, BC V1V 1W5 
Statistical Analyses 
Descriptive statistics (means, SD, and frequencies) will be calculated. Significance 
will be set at P < 0.05. Data across time will be analyzed in R using a linear mixed 
effects model with subjects as a random factor and condition and time as fixed 
factors. Assumptions will be tested using Q-Q plots of residuals. Data with skewed 
distributions will be log-transformed or non-linear mixed effects models will be 
used. All participants will be included in the intention-to-treat (ITT) analyses and 
missing data will not be imputed, per contemporary guidelines with use of linear 
mixed models [10].  
 
Sample Size 
We will recruit 12 participants to be included in this cross-over study, which has 
blood β-OHB (as represented by the area under the curve across the 4 hours 
following ingestion of the supplement) as the primary outcome. A previous 
pharmacokinetic study exploring the effects of a ketone monoester provided at a 
dose of ~10 g in healthy adults found that blood β-OHB was raised to 1.4 ± 0.6 mM 
within 30 minutes, and to an average of 0.5 ± 0.5 mM across the entire 4-hour 
post-supplementation period [2], which represents an effect size of d > 1.0 
compared to baseline. Using the outcome data from this study (N = 15) and 
assuming 80% power with an alpha level of 0.05 and a moderate correlation 
between repeated measures of 0.5, a total sample size of N = 6 would be sufficient 
to detect an increase in average blood β-OHB compared to baseline (calculated 
using G*Power v3.1). In order to allow for a more nuanced evaluation of 
differences between ketone supplements and to account for any dropouts or 
missing blood samples, we will recruit 12 participants (aiming for equal males and 
females), which is in line with the sample size of previous pharmacokinetic studies 
of ketone supplements [2]. 

 
 
 
The Effect of Different Ketone Supplements on Blood β-OHB and Blood 
Glucose in Healthy Individuals 
 
Version 2 – Feb 15, 2022 [H22-00083] – NCT05273411 
 
9
Faculty of Health and Social Development 
School of Health and Exercise Sciences 
3333 University Way 
Kelowna, BC V1V 1W5 
 
Significance 
The results of this study will help determine the effect of different types of ketone 
supplements on blood β-OHB and glucose. This is a pilot study and it will provide 
useful data to better answer future research questions and aid in the design of 
longer-term studies.  
 
Outcome Measurements and Methods 
The primary outcome will be total β-OHB as represented by the area under the 
curve across 4 hours. Secondary outcomes include blood glucose, blood pressure, 
heart rate, hunger and fullness, gastrointestinal discomfort, and taste and 
supplement acceptability. 
 
Anthropometric Measures 
Waist circumference (cm), height (cm), and weight (kg) will be measured using 
standard procedures. Body mass index (BMI) will be calculated as kg/m2.  
 
Physiological Measures  
Resting blood pressure and heart rate will be collected for descriptive purpose and 
throughout the study visits (at the same time points as blood samples are 
collected). Before measurements, participants will be instructed to sit quietly and 
rest for 5 minutes. A finger tip heart rate monitor will track heart rate. Blood 
pressure will be collected using a cuff around the upper arm, a stethoscope will be 
placed in the cubital fossa for systolic and diastolic blood pressure.   

 
 
 
The Effect of Different Ketone Supplements on Blood β-OHB and Blood 
Glucose in Healthy Individuals 
 
Version 2 – Feb 15, 2022 [H22-00083] – NCT05273411 
 
10
Faculty of Health and Social Development 
School of Health and Exercise Sciences 
3333 University Way 
Kelowna, BC V1V 1W5 
 
Blood Sampling 
 
Blood samples will be collected via finger prick. Blood β-OHB and glucose levels 
will be measured using the “Keto-Mojo” handheld monitor (which we have used 
extensively in our previous studies) at baseline and 15, 30, 60, 90, 120, 180, and 
240 minutes after ingestion of the ketone supplement. 
 
 
 
 
 

 
 
 
The Effect of Different Ketone Supplements on Blood β-OHB and Blood 
Glucose in Healthy Individuals 
 
Version 2 – Feb 15, 2022 [H22-00083] – NCT05273411 
 
11
Faculty of Health and Social Development 
School of Health and Exercise Sciences 
3333 University Way 
Kelowna, BC V1V 1W5 
References 
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Physiol 2017;8:848. 
3. Greaves G, Xiang R, Rafiei H, Malas A, Little JP. Prior ingestion of a ketone monoester 
supplement reduces postprandial glycemic responses in young healthy-weight individuals. Appl 
Physiol Nutr Metab 2021;46(4):309-317. 
4. Myette-Côté É, Neudorf H, Rafiei H, Clarke K, Little JP. Prior ingestion of exogenous ketone 
monoester attenuates the glycaemic response to an oral glucose tolerance test in healthy young 
individuals. J Physiol 2018;596(8):1385-1395.  
5. Myette-Côté É, Caldwell HG, Ainslie PN, Clarke K, Little JP. A ketone monoester drink reduces 
the glycemic response to an oral glucose challenge in individuals with obesity: a randomized trial. 
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7. Walsh JJ, Myette-Côté É, Neudorf H, Little JP. Potential therapeutic effects of exogenous ketone 
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9. Diamond MP, Simonson DC, and DeFronzo RA. Menstrual cyclicity has a profound effect on 
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10. Chakraborty H. A mixed model approach for intent-to-treat analysis in longitudinal clinical trials 
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approach-intent-treat-analysis-longitudinal-clinical-trials-missing-values 
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