The Effects of Exogenous Beta-Hydroxybutyrate Supplementation on Metrics of Safety and Health

Abstract

The ketogenic diet is a high-fat, very low-carbohydrate, moderate-protein diet that will induce a state of ketosis. Ketosis is a metabolic state characterized by elevated ketone body production in response to the absence of carbohydrates. Some drawbacks of the ketogenic diet are that it can be difficult to adhere to due to its restrictive nature, and it can also cause some undesirable side effects like gastrointestinal distress and increases in apoB-lipoproteins. In order to maximize the benefit of ketosis and to minimize side effects, supplementing with exogenous beta-hydroxybutyrate may induce a state of temporary ketosis without undesirable side effects. In the present study, 22 healthy male and female adults consumed 12.75 grams of beta-hydroxybutyrate salts or maltodextrin placebo twice daily for 90 days. Comprehensive blood safety analysis, body composition, bone densitometry, psychological and immune surveys, and blood pressure were administered at baseline, 30, 60, and 90 days. There were no significant differences in any measures collected, indicating that exogenous beta-hydroxybutyrate had no detrimental impact on fasting blood values such as electrolyte levels, glucose, hemoglobin A1c, complete blood count, body composition, bone density, psychological well-being, immune status, or blood pressure. We conclude that supplementing with exogenous beta-hydroxybutyrate is safe and well-tolerated by healthy adults.

Full text

International Journal of Nutrition and Food Sciences
2020; 9(6): 154-162
http://www.sciencepublishinggroup.com/j/ijnfs
doi: 10.11648/j.ijnfs.20200906.13
ISSN: 2327-2694 (Print); ISSN: 2327-2716 (Online)
The Effects of Exogenous Beta-Hydroxybutyrate
Supplementation on Metrics of Safety and Health
Matthew Stefan
*
, Matthew Sharp, Raad Gheith, Ryan Lowery, Jacob Wilson
Research Department, The Applied Science and Performance Institute, Tampa, Florida, USA
Email address:
*
Corresponding author
To cite this article:
Matthew Stefan, Matthew Sharp, Raad Gheith, Ryan Lowery, Jacob Wilson. The Effects of Exogenous Beta-Hydroxybutyrate Supplementation
on Metrics of Safety and Health. International Journal of Nutrition and Food Sciences. Vo l . 9 , N o . 6 , 2 0 2 0 , p p . 1 5 4 - 1 6 2 .
doi: 10.11648/j.ijnfs.20200906.13
Received: November 9, 2020; Accepted: November 24, 2020; Published: December 8, 2020
Abstract:
The ketogenic diet is a high-fat, very low-carbohydrate, moderate-protein diet that will induce a state of ketosis.
Ketosis is a metabolic state characterized by elevated ketone body production in response to the absence of carbohydrates. Some
drawbacks of the ketogenic diet are that it can be difficult to adhere to due to its restrictive nature, and it can also cause some
undesirable side effects like gastrointestinal distress and increases in apoB-lipoproteins. In order to maximize the benefit of
ketosis and to minimize side effects, supplementing with exogenous beta-hydroxybutyrate may induce a state of temporary
ketosis without undesirable side effects. In the present study, 22 healthy male and female adults consumed 12.75 grams of
beta-hydroxybutyrate salts or maltodextrin placebo twice daily for 90 days. Comprehensive blood safety analysis, body
composition, bone densitometry, psychological and immune surveys, and blood pressure were administered at baseline, 30, 60,
and 90 days. There were no significant differences in any measures collected, indicating that exogenous beta-hydroxybutyrate
had no detrimental impact on fasting blood values such as electrolyte levels, glucose, hemoglobin A1c, complete blood count,
body composition, bone density, psychological well-being, immune status, or blood pressure. We conclude that supplementing
with exogenous beta-hydroxybutyrate is safe and well-tolerated by healthy adults.
Keywords:
Beta-Hydroxybutyrate, Ketosis, Safety, Exogenous Ketones
1. Introduction
The ketogenic diet is categorized as a high-fat, very-low
carbohydrate, and moderate protein dietary strategy that is
meant to mimic a fasted state by restricting carbohydrate
intake. Research has commonly defined the intakes of a
ketogenic diet as less than 50 grams of carbohydrates per day,
or 5 to 10 percent of total caloric contribution coming from
carbohydrates, with fat contributing up to 90 percent of total
caloric intake [1, 2]. The goal of the ketogenic diet is to induce
ketosis – a metabolic state characterized by increased ketone
body production in response to the absence of carbohydrates.
In order to reach a state of nutritional ketosis, blood ketone
concentration should be between 0.5 millimolar (mM) and 3.0
mM [3]. This rise in endogenous ketones is dependent on
macronutrient availability of glucose and fatty acids, and the
hormonal signaling of glucagon, insulin, and cortisol.
There are many benefits to human health for being in a state
of ketosis from consuming a ketogenic diet. Weight loss
occurs due to the reliance on fatty acid storage, and from the
mitochondria regaining their metabolic flexibility (countering
insulin resistance) [4, 5]. Young et al. demonstrated that as
ketone levels rose, fat loss rose as well [6]. In addition to
weight loss, research has shown that the ketogenic diet does
not have a negative impact on hunger hormones despite a
decline in total caloric intake [7]. To achieve an appetite
suppressive effect, ketones concentrations only need to reach
mild ketosis (greater than 0.5 mM) [8, 10]. Research has also
demonstrated that the application of the ketogenic diet can
have therapeutic benefits on diseases that impact metabolism
[9]; reduce the incidence of seizures in children with epilepsy
[11], improve outcomes of certain neurogenerative diseases
like Parkinson’s Disease [12], may help control glycolytic
phenotype of various cancers by limiting glucose availability

International Journal of Nutrition and Food Sciences 2020; 9(6): 154-162 155
[13], and lower glucose and hemoglobin A1c concentrations
in individuals with type 2 diabetes [14, 15]. Lastly, e le vate d
blood ketones could improve endurance performance and
further optimize substrate metabolism by providing an
alternative source for oxidative phosphorylation [16, 17].
In addition to being carbohydrate restrictive, adherence to
the ketogenic diet can be difficult due to some undesirable side
effects like gastrointestinal discomfort [18] and increases in
apoB-lipoproteins [19]. Therefore, temporary and rapid rises
in blood ketone concentrations with no dietary changes may
be of potential interest and benefit [20]; hence, the relevance
of exogenous ketones [21-23]. The safety of ketone esters has
been previously explored, however, there is a void in the
literature on the safety of ketone salts, which is what this study
investigated. One previous study on the safety of ketone salts
demonstrated that two servings of 7 grams of
beta-hydroxybutyrate (BHB) combined with erythritol,
L-Taurine, and L-Leucine was safe as demonstrated by no
changes in complete blood count (CBC) or biomarkers of a
comprehensive metabolic panel over 6 weeks [24]. Moreover,
markers of cardiovascular health, such as blood pressure,
improved while heart rate remained unchanged. The purpose
of this study is to extend this research to 90 days with a dosage
of 12.75 grams twice per day with additional metrics of safety
and health.
2. Methods
2.1. Subject Criteria
Twenty-two healthy male and female subjects aged 18 to 50
years old enrolled for study participation. Exclusion criteria
included: hypertension, obesity (body mass index [BMI] >30
kg/m
2
), smoking or using smokeless tobacco, taking any
prescription medication, or having any underlying health
conditions (metabolic, heart disease, diabetes, kidney disease).
This study was approved by an external institutional review
board (Integ Review IRB, Austin, TX, USA) and all
procedures were in agreement with institutional guidelines and
the Declaration of Helsinki. Prior to engagement in any study
procedures, subjects provided written informed content.
Tab l e 1. Descriptive Subject Characteristics.
BHB PLA
Sample Size (n=11) (n=11)
Age (years) 44.45 ± 7.30 45.55 ± 9.05
Height (cm) 166.49 ± 9.80 169.03 ± 11.15
Wei g ht ( k g) 72.79 ± 14.67 78.81 ± 18.07
Body Mass Index (kg/m
2
) 26.32 ± 5.13 27.27 ± 3.66
2.2. Study Design
The study design was a randomized, double-blinded,
placebo-controlled trial. Subjects were stratified into quartiles
based on BMI and subjects from each quartile were randomly
assigned to conditions using a random number generator
(random. org). The conditions were sent to the primary
investigator in white packages labeled “A” or “B”. These were
administered as 12.75-gram servings of a R-Beta
Hydroxybutyrate (BHB) salt blend (KetoNAT™; Science
Backed Solutions, LLC; Melissa, TX, USA) or a similarly
flavored iso-energetic, iso-volumetric maltodextrin placebo
twice daily for 90 days for a total of 25.5-grams of the
respective condition, daily. Subjects underwent baseline
testing (PRE) which included: blood draw for safety measures
(complete blood count, comprehensive metabolic panel,
automated differential, and hemoglobin A1c), resting blood
pressure and heart rate, psychological mood assessment
(Profile of Mood States; [POMS]), immune status
questionnaire, body composition and bone densitometry.
Following PRE testing, subjects were given a 30-day supply
of either condition “A” or condition “B”. Subjects were
instructed to consume one serving in the morning and one
serving in the afternoon with at least three hours of separation
between servings. Subjects were also asked to track their
caloric intake 3 days every week for the duration of the study.
Lastly, subjects submitted VAS (visual analog scales) to report
subjective measures of satiety, hunger, and psychological
feelings (well-being, mental clarity, etc.). Testing was
repeated for all study procedures in an identical manner to
PRE at 30 days, 60 days, and 90 days following the original
PRE testing date, with the exception of the DXA which was
only performed at PRE and 90 days. Study procedures are
further described below.
2.3. Bone Densitometry and Body-Composition Analysis
Bone densitometry and body composition was determined
by a whole-body scan on a dual-energy x-ray absorptiometry
device (Horizon A DXA System, Hologic Inc, Marlborough,
MA, USA). Fat-free mass, fat mass, body fat percentage, bone
mineral content, and bone density was determined for the total
body with the subject lying in a supine position with knees and
elbows extended. Subjects were instructed not to move for the
entire duration of the scan (approximately 5 minutes). Results
from each scan were uploaded and accessed on computer that
was directly linked to the DXA device. Calibration of the
DXA device was done against a phantom provided by the
manufacturing company prior to testing.
2.4. Venous Blood Measures
Ve no us b l oo d w as e x tr a ct ed by v e ni pu n ct u re o f t he a ntecubital
vein using a 21-gauge syringe and collected into a 10mL EDTA
vacutainer tube (BD Vacutainer®, Becton, Dickinson and
Company, Franklin Lakes, NJ, USA) by a certified phlebotomist.
Afterward, blood samples were centrifuged at 2500 rpm for 10
minutes at 4°C. Resulting serum samples were then aliquoted and
stored at −80°C until further analysis. Samples were thawed once
and analyzed in duplicate in the same assay for each analysis to
avoid compounded inter-assay variance.
2.5. Blood Pressure and Heart Rate
Subjects rested in a supine position for 5 min in a quiet room
at 228°C before the baseline hemodynamic measurements were
obtained. Resting brachial blood pressure and heart rate were
measured on the right arm with an automated digital

156 Matthew Stefan et al.: The Effects of Exogenous Beta-Hydroxybutyrate Supplementation on Metrics of Safety and Health
oscillometric sphygmomanometer (Omron, Model HEM
705-CP; Omron Corporation, Shimogyo-ku, Kyoto, Japan).
Three readings separated by 1-min intervals were taken, and the
mean was used for the analysis.
2.6. Immune Status Questionnaire (ISQ) and Profile of
Mood States (POMS)
The Immune Status Questionnaire (ISQ) is a validated
self-assessment of subjective values of seven different
common symptoms associated with disease [25]. The ISQ was
scored on a 5-point Likert scale from 0 to 4 for how often the
subject has had the following symptoms in the past week;
Never, Sometimes, Regularly, Often, and Almost Always. The
values were summed up to equal a raw score. The raw score
was then converted into a final score between zero and ten,
with 0 being the poor immune status, and ten being excellent
immune status [26].
The Profile of Mood States is a validated self-assessment of
subjective values of forty different moods [25]. Those moods
then fall into seven categories: Tension, Anger, Fatigue,
Depression, Esteem - Related, Vigor, and Confusion. Subjects
were asked to assess each of the forty moods, and if they are
feeling that particular mood “right now”. Subjects assessed the
moods according to a 5-point Likert scale from 0 to 4: Not at All,
A Little, Moderately, Quite a Lot, Extremely. The following
formula was used to determine the overall POMS score:
(Tension + Depression + Anger + Fatigue + Confusion) –
(Vigor + Esteem-Related) + 100
A lo wer scor e indi cate d a be tter mo od , whi le a h igher score
indicated a poor mood [24].
2.7. Visual Analog Scales for Perceived Hunger and
Perceived Mental Clarity
The perceptual measures collected for the study were
perceived Hunger and perceived Mental Clarity. Hunger and
Mental Clarity scales consisted of a scalar representation
numbering from 0-10. On the Hunger Scale, visual descriptors
of “not hungry”, “adequately hungry” and “very hungry”
presented at numbers 0, 5, and 10, respectively. On the Mental
Clarity scale, visual descriptors of “Poor Mental Clarity”,
“Adequate Mental Clarity”, and “Very Mentally Clear” are
presented at numbers 0, 5, and 10, respectively.
2.8. Calorie and Macronutrient Reporting
Subjects were asked to record, and then report, their caloric
intake three times per week using a mobile tracking
application (MyFitnessPal, San Francisco, CA, USA).
2.9. Statistical Analysis
All statistical analyses were performed at the completion of
the study using GraphPad Prism (Version 8, San Diego, CA,
USA). Dependent variables were assessed for normality
(Shapiro-Wilk test) and homogeneity of variances (Levene’s
test). Two-way mixed model analysis of variance (ANOVA)
was performed assuming group and time as fixed factors and
subjects as a random factor. Whenever a significant F value
was obtained, a post hoc test with a Bonferroni adjustment
was used to for multiple comparisons purposes. The alpha
level was set a p ≤ 0.05. Data are reported as mean ± standard
deviation.
3. Results
3.1. Complete Blood Count
There was no significant between or within group
differences in Complete Blood Count values (p > 0.05, Table
2). Mean and standard deviation are displayed in Table 2.
Tab l e 2. Complete Blood Count Results.
PRE 30 DAY 60 DAY 90 DAYS p Value
WBC (K/uL)
BHB 5.13 ± 0.96 5.07 ± 0.95 5.38 ± 1.07 6.62 ± 2.49 0.1001
PLA 5.91 ± 1.03 5.91 ± 1.05 5.94 ± 1.03 6.19 ± 1.06
RBC (M/uL)
BHB 4.66 ± 0.41 4.61 ± 0.33 4.67 ± 0.34 4.70 ± 0.39 0.1084
PLA 4.36 ± 0.25 4.44 ± 0.34 4.37 ± 0.38 4.31 ± 0.40
Hemoglobin (g/dL)
BHB 14.43 ± 1.06 14.28 ± 0.85 14.37 ± 0.89 14.45 ± 1.01 0.1203
PLA 13.40 ± 1.25 13.64 ± 1.22 13.31 ± 1.29 13.15 ± 1.46
Hematocrit (%)
BHB 47.64 ± 3.07 46.82 ± 2.47 47.17 ± 2.76 46.92 ± 3.24 0.1233
PLA 44.44 ± 3.05 45.05 ± 3.23 45.05 ± 3.23 44.05 ± 3.45
MCV (fl)
BHB 102.36 ± 3.41 101.55 ± 3.53 101.00 ± 3.66 100.00 ± 3.29 0.8246
PLA 101.91 ± 6.69 101.64 ± 6.09 101.09 ± 6.27 100 ± 7.17
MCH (pg)
BHB 31.01 ± 1.01 31.00 ± 1.39 30.85 ± 1.11 30.85 ± 1.07 0.9716
PLA 30.73 ± 2.60 30.85 ± 2.76 30.60 ± 2.53 30.59 ± 2.81
MCHC (g/dL)
BHB 30.29 ± 0.46 30.50 ± 0.66 30.52 ± 0.79 30.85 ± 0.71 0.9812
PLA 30.10 ± 0.95 30.26 ± 1.08 30.24 ± 0.90 30.55 ± 1.06
RDW (%)

International Journal of Nutrition and Food Sciences 2020; 9(6): 154-162 157
PRE 30 DAY 60 DAY 90 DAYS p Value
BHB 13.15 ± 0.59 13.14 ± 0.57 12.94 ± 0.43 12.96 ± 0.47 0.4562
PLA 12.73 ±3.26 13.30 ± 1.06 13.30 ± 1.04 13.50 ± 0.42
Platelets (k/uL)
BHB 218.64±5.86 230.73±70.03 232.27±65.23 236.73±79.15 0.4181
PLA 218.09±34.31 217.5±33.87 217.64±40.66 222.00±45.73
Data reported in mean and standard deviation. P-value is from group by time interaction effect.
3.2. Automated Differential
There was no significant between or within group differences in any values of Automated Differential Cell Count (p > 0.05,
Tab le 3). Mean an d stand ard d ev iatio n are d ispla yed in Table 3.
Tab l e 3. Automated Differential Cell Count
PRE 30 DAY 60 DAY 90 DAYS p Value
Lymphocytes (%)
BHB 35.77 ± 8.38 35.34 ± 7.83 35.68 ± 8.83 30.99 ± 10.14 0.2397
PLA 35.86 ± 7.02 38.42 ± 7.16 37.58 ± 6.68 37.25 ± 6.28
Monocytes (%)
BHB 8.17 ± 5.42 6.01 ± 2.38 5.81 ± 2.45 6.70 ± 3.96 0.4749
PLA 6.35 ± 1.79 6.21 ± 2.10 5.90 ± 1.81 5.72 ± 1.73
Eosionophil (%)
BHB 2.02 ± 1.28 2.26 ± 1.32 2.35 ± 1.34 2.17 ± 1.35 0.6470
PLA 2.01 ± 1.21 2.02 ± 1.34 1.92 ± 1.16 2.05 ± 1.02
Basophil (%)
BHB 1.34 ± 0.27 1.22 ± 0.40 1.09 ± 0.32 1.05 ± 0.23 0.4171
PLA 1.15 ± 0.43 1.14 ± 0.46 1.03 ± 0.35 1.13 ± 0.74
Granulocytes (%)
BHB 54.76 ± 8.55 55.16 ± 8.82 54.15 ± 9.98 59.03 ± 12.18 0.4594
PLA 54.63 ± 8.75 52.25 ± 6.94 53.38 ± 7.07 53.98 ± 7.24
Lymphocytes (k/uL)
BHB 2.03 ± 0.62 1.76 ± 0.31 1.93 ± 0.37 1.86 ± 0.40 0.2579
PLA 2.13 ± 0.53 2.27 ± 0.58 2.23 ± 0.46 2.29 ± 0.53
Monocytes (k/uL)
BHB 0.33 ± 0.15 0.28 ± 0.11 0.30 ± 0.10 0.41 ± 0.25 0.0812
PLA 0.38 ± 0.08 0.35 ± 0.10 0.35 ± 0.12 0.34 ± 0.10
Eosionophil (k/uL)
BHB 0.09 ± 0.05 0.12 ± 0.06 0.12 ± 0.06 0.14 ± 0.08 0.1565
PLA 0.13 ± 0.05 0.13 ± 0.06 0.12 ± 0.08 0.13 ± 0.06
Basos (k/uL)
BHB 0.09 ± 0.03 0.07 ± 0.05 0.06 ± 0.05 0.08 ± 0.04 0.4746
PLA 0.07 ± 0.05 0.08 ± 0.04 0.07 ± 0.05 0.08 ± 0.06
Granulocytes (k/uL)
BHB 2.60 ± 0.99 2.85 ± 0.89 2.98 ± 0.94 4.15 ± 2.62 0.1234
PLA 3.26 ± 0.84 3.11 ± 0.77 3.21 ± 0.87 3.35 ± 0.81
Data reported in mean and standard deviation. P-value is from group by time interaction effect.
3.3. Comprehensive Metabolic Panel
There was no significant between or within group differences in any values of the comprehensive metabolic panel (p > 0.05,
Tab le 4). Mean an d stand ard d ev iatio n are d ispla yed in Table 4.
Tab l e 4. Comprehensive Metabolic Panel
PRE 30 DAY 60 DAY 90 DAY p Value
Total Protein (g/dL)
BHB 6.65 ± 0.35 6.48 ± 0.28 6.51 ± 0.38 6.48 ± 0.43 0.5227
PLA 6.58 ± 0.41 6.56 ± 0.44 6.47 ± 0.36 6.42 ± 0.38
Albumin (g/dL)
BHB 4.49 ± 0.22 4.28 ± 0.22 4.28 ± 0.22 4.44 ± 0.28 0.4284

158 Matthew Stefan et al.: The Effects of Exogenous Beta-Hydroxybutyrate Supplementation on Metrics of Safety and Health
PRE 30 DAY 60 DAY 90 DAY p Value
PLA 4.40 ± 0.20 4.29 ± 0.25 4.2 ± 0.20 4.29 ± 0.29
Globulin (g/dL)
BHB 2.15 ± 0.05 2.2 ± 0.18 2.25 ± 0.26 2.05 ± 0.42 0.8957
PLA 2.18 ± 0.27 2.27 ± 0.23 2.27 ± 0.21 2.13 ± 0.26
ALB: GLOB (U/L)
BHB 2.10 ± 0.24 1.97 ± 0.17 1.97 ± 0.17 1.91 ± 0.23 0.4320
PLA 2.05 ± 0.23 1.89 ± 0.16 1.88 ± 0.17 2.05 ± 0.30
Bilirubin (mg/dL)
BHB 0.66 ± 0.21 0.63 ± 0.11 0.65 ± 0.22 0.62 ± 0.17 0.8334
PLA 0.57 ± 0.21 0.55 ± 0.14 0.63 ± 0.21 0.54 ± 0.17
Alkaline Phosphate (U/L)
BHB 52.73 ± 11.42 50.09 ± 13.48 49.91 ± 13.23 50.09 ± 11.69 0.4123
PLA 52.64 ± 14.70 51.82 ± 15.09 52.45 ± 12.23 54.09 ± 12.93
AST (U/L)
BHB 23.27 ± 6.23 27.73 ± 19.89 24.82 ± 9.59 23.55 ± 10.47 0.3370
PLA 28.27 ± 16.87 23.64 ± 8.61 24.45 ± 7.53 22.64 ± 7.70
ALT (U/L)
BHB 23.09 ± 9.60 26.36 ± 12.92 26.09 ± 16.16 25.64 ± 17.47 0.2445
PLA 29.00 ± 27.07 23.27 ± 9.71 25.73 ± 15.11 24.82 ± 15.68
BUN (mg/dL)
BHB 17.00 ± 4.27 14.73 ± 3.17 15.36 ± 4.11 16.09 ± 3.96 0.1065
PLA 15.91 ± 3.75 17.64 ± 5.41 17.00 ± 6.23 17.36 ± 6.34
Creatinine (mg/dL)
BHB 0.86 ± 0.22 0.90 ± 0.23 0.92 ± 0.22 0.89 ± 0.23 0.3388
PLA 0.85 ± 0.13 0.91 ± 0.13 0.89 ± 0.16 1.58 ± 2.17
BUN/Creatinine (mg/dL)
BHB 24.23 ± 4.84 21.30 ± 0.14 21.40 ± NA 21.40 ± N/A 0.0881
PLA 22.48 ± 0.86 24.73 ± 2.11 25.23 ± 1.27 23.30 ± N/A
eGFR (mL/min)
BHB 88.73 ± 15.41 84.55 ± 15.71 81.36 ± 11.93 84.82 ± 12.96 0.2965
PLA 86.64 ± 9.01 80.73 ± 10.07 82.36 ± 11.53 79.00 ± 9.34
Sodium (mg/dL)
BHB 141.45 ± 2.21 140.91 ± 1.64 140.18 ± 1.54 139.64 ± 2.06 0.6043
PLA 141.18 ± 1.83 140.45 ± 1.21 140.00 ± 1.73 140.18 ± 2.09
Potassium (mg/dL)
BHB 4.35 ± 0.35 4.27 ± 0.35 4.13 ± 0.23 4.26 ± 0.24 0.4547
PLA 4.35 ± 0.34 4.19 ± 0.18 4.08 ± 0.16 4.07 ± 0.18
Chloride (mg/dL)
BHB 103.55 ± 2.34 102 ± 1.95 102.91 ± 1.81 102.91 ± 2.26 0.9183
PLA 105.36 ± 2.46 103.82 ± 1.47 104.18 ± 1.78 104.55 ± 2.46
Carbon Dioxide (mL/min)
BHB 27.55 ± 2.25 29.36 ± 1.57 28.91 ± 1.58 29.36 ± 2.42 0.1070
PLA 26.55 ± 2.07 26.64 ± 2.11 27.18 ± 1.99 27.00 ± 2.10
Calcium (mg/dL)
BHB 9.23 ± 0.31 8.97 ± 0.35 9.21 ± 0.35 9.19 ± 0.28 0.1681
PLA 9.14 ± 0.34 8.95 ± 0.32 9.05 ± 0.36 8.92 ± 0.37
Glucose (mg/dL)
BHB 73.45 ± 8.00 87.64 ± 4.43 87.27 ± 4.98 93.36 ± 8.88 0.7419
PLA 76.36 ± 12.47 89.73 ± 10.25 92.09 ± 9.02 93.36 ± 10.53
Hemoglobin A1c
BHB 5.25 ± 0.27 5.10 ± 0.30 5.20 ± 0.31 5.38 ± 0.26 0.3925
PLA 5.32 ± 0.25 5.25 ± 0.29 5.38 ± 0.30 5.48 ± 0.30
Data reported in mean and standard deviation. P-value is from group by time interaction effect. (ALB:GLOB = Albumin:Globulin Ratio, AST =aspartate
aminotransferase, ALT = alanine transaminase, BUN = blood urea nitrogen, eGFR = estimated glomerular filtration rate.)
3.4. Blood Pressure and Heart Rate
There was no significant between or within group differences in resting blood pressure or heart rate (p > 0.05, Table 5). Mean
and standard deviation are displayed in Table 5.

International Journal of Nutrition and Food Sciences 2020; 9(6): 154-162 159
Tab l e 5. Blood Pressure and Cardiovascular Results.
PRE 30 DAY 60 DAY 90 DAYS p Value
Systolic BP (mmHg)
BHB 113.00±10.25 112.18±9.66 109.00±11.25 109.09 ± 9.27 0.6480
PLA 115.91±11.38 117.45±10.35 116.64±10.60 115.00 ± 8.56
Diastolic BP (mmHg)
BHB 68.27 ± 10.68 64.36 ± 7.12 63.55 ± 10.00 65.45 ± 7.61 0.5610
PLA 70.82 ± 10.25 68.55 ± 11.85 70.55 ± 11.10 68.36 ± 8.54
Heart Rate (bpm)
BHB 62.55 ± 10.68 64.55 ± 7.03 65.00 ± 10.00 63.82 ± 8.15 0.4459
PLA 61.64 ± 8.32 64.73 ± 8.68 61.64 ± 8.41 60.91 ± 8.60
Data reported in mean and standard deviation. P-value is from group by time interaction effect. (BP = blood pressure).
3.5. Profile of Mood States (POMS) & Immune Status Questionnaire (ISQ)
There was no significant between or within group differences for responses to the POMS questionnaire or the ISQ (p > 0.05,
Tab le 6). Mean an d stand ard d ev iatio n are d ispla yed in Table 6.
Tab l e 6. Survey Results
PRE 30 DAYS 60 DAYS 90 DAYS p Value
Total POMS Score (a.u.)
BHB 82.55 ± 17.06 77.36 ± 13.43 79.55 ± 12.13 86.09 ± 9.97 0.7240
PLA 73.64 ± 9.14 73.91 ± 11.22 74.45 ± 10.82 78.09 ± 7.83
ISQ Total Score (a.u. )
BHB 9.00 ± 1.10 8.73 ± 1.74 9.00 ± 1.48 8.73 ± 1.42 0.2195
PLA 9.27 ± 0.47 9.45 ± 0.52 9.36 ± 0.67 9.64 ± 0.50
Data reported in mean and standard deviation. P-value is from group by time interaction effect.
3.6. Body Composition & Bone Densitometry
There was no significant between or within group differences in any body composition or bone densitometry values (p > 0.05,
Tab le 7). Mean an d stand ard d ev iatio n are d ispla yed in Table 7.
Tab l e 7. Body Composition & Bone Densitometry Results.
PRE DAY 90 p Value
Total Mass (kg)
BHB 72.66 ± 14.24 72.80 ± 14.44 0.9727
PLA 79.53 ± 17.98 79.60 ± 18.92
Fat Ma ss (kg)
BHB 23.21 ± 9.55 23.96 ± 10.48 0.8739
PLA 23.74 ± 6.87 25.31 ± 8.26
Fat Free Mass (kg)
BHB 49.45 ± 10.96 48.84 ± 9.69 0.5330
PLA 55.79 ± 14.28 54.28 ± 12.66
Body Fat %
BHB 31.66 ± 9.44 32.29 ± 9.63 0.3223
PLA 29.96 ± 5.94 31.45 ± 5.90
Bone Mineral Density (g/cm
2
)
BHB 1.13 ± 0.11 1.11 ± 0.09 0.1530
PLA 1.16 ± 0.13 1.18 ± 0.11
Bone Mineral Content (g)
BHB 2291.19 ± 369.46 2248.45 ± 245.24 0.2638
PLA 2510.98 ± 501.73 2534.13 ± 409.74
Data reported in mean and standard deviation. P-value is from group by time interaction effect.
4. Discussion
In this study, we demonstrated the safety of exogenous
BHB under uncontrolled conditions of daily living. The most
significant finding of this study was that sustained 25.5 grams
of daily exogenous ketone salt consumption for 90 days was
safe for healthy adults and that it had no adverse effect on any
blood health markers, hemoglobin A1c, psychological
well-being, or cardiovascular markers of health.
Comprehensive metabolic panel, complete blood count, and
automated differential cell count remained normal and
unaltered after supplementing twice daily with exogenous

160 Matthew Stefan et al.: The Effects of Exogenous Beta-Hydroxybutyrate Supplementation on Metrics of Safety and Health
BHB for 90 days. Furthermore, there were no significant
changes in the POMS or ISQ, resting blood pressure, or
resting heart rate. The findings in this study support, and
further build upon, a previous study by Holland et al. [24]
demonstrating that 6 weeks of exogenous ketone salts
supplementation did not negatively impact various markers of
human health and safety in adults.
There are many controversial views regarding the ketogenic
diet, ketosis, and exogenous ketones. Two such views are that
ketosis can increase the risk of complications in the liver [27],
and the kidneys [28]. However, in human studies, it was
demonstrated that the ketogenic diet may improve clinical
outcomes of nonalcoholic fatty liver disease [29, 31]. In the
present study, we found that markers of liver health; total
protein, albumin, globulin, ALB:GLOB ratio, bilirubin,
alkaline phosphate, AST, and ALT; were unaffected and no
different from placebo with exogenous ketone
supplementation in a healthy population over 90 days.
Depending on macronutrient distribution of caloric intake,
the ketogenic diet can be considered a high protein diet (≥ 20%
of caloric intake). It has been posited that diets higher in protein
could lower pH and increase the acidic load on the kidneys [28].
However, the previously referenced study used a high- protein,
low-carbohydrate diet, while a typical ketogenic diet consists of
low to moderate protein and very-low carbohydrate [30]. With a
typical ketogenic diet, Kossof et al. [32] demonstrated no
increased risk of kidney stones in children. Our study
demonstrated no changes in kidney function markers such as
BUN, creatinine, BUN/creatinine ratio, and eGFR in a healthy
population over 90 days. In addition, acid-base balance was
maintained as demonstrated by blood carbon dioxide and
chloride levels.
The ketogenic diet has shown to lead to calcium loss, and in
some cases, can increase the risk of bone loss. Previous
research has suggested that the ketogenic diet can increase
calcium excretion, which can lead to bone mass loss in
children and adolescents [33–35]. Our study demonstrated no
blood calcium or electrolyte loss, as all electrolyte levels were
unchanged over 90 days in a healthy adult population.
Moreover, we found no changes in bone mineral density or
bone mineral content as assessed by whole-body DXA scans
at PRE and Day 90. These results suggest that BHB does not
lead to bone density or bone mineral content loss.
Lastly, it has been postulated that electrolytes may alter
different markers of cardiovascular health such as blood
pressure and heart rate [36]. Exogenous ketone salts, like the
ones used in the present study, are bound to calcium and
magnesium in order to improve transport and absorption
across the gut-blood barrier. Therefore, it is reasonable to
investigate if altering dietary intake of electrolytes with the
consumption of exogenous ketone salts may have an effect on
blood pressure. However, in our present study, systolic blood
pressure, diastolic blood pressure, and heart rate were
unaffected in the resting state. In addition, blood
concentrations of sodium, potassium, chloride, and calcium all
remained unaltered.
A l im ita ti on o f t he p re sent st ud y i s th e me th od o f
supplementation, and the lack of exercise and dietary control.
Subjects were provided the supplement every 30 days when
they came into the laboratory for testing. They were asked to
return any unused supplements to the laboratory to help keep
subjects honest and to track adherence. Lastly, diet and
exercise were not controlled. However, to keep subjects
accountable, they were encouraged to track and report dietary
intake via a mobile application. Dietary records demonstrated
no differences in daily average of total calories consumed
(BHB: 1430.71 ± 370.84 vs PLA: 1560.04 ± 394.54 kcal/day,
p=0.4381). Future research may seek to directly compare the
effects of exogenous ketone esters and exogenous ketone salts.
5. Conclusion
Exogenous ketone salt supplementation (BHB) can be
considered safe and well tolerated. BHB showed no changes
in comprehensive metabolic panel, automated differential cell
count, complete blood count, hemoglobin A1c, resting blood
pressure and heart rate or psychological surveys over 90 days
of supplementation in a healthy population when compared to
PLA. This study established the safety of long term BHB
supplementation, but further investigation is needed to
examine the efficacy of exogenous ketone supplementation in
other areas of health, longevity, cognitive function, and other
chronic conditions.
Funding
Pruvit Ventures Inc. (Melissa, Texas USA) financially
supported the study and supplied the treatment and control
conditions used in this investigation.
Disclosures
Jacob Wilson and Ryan Lowery receive honorira from
Pruvit Ventures Inc. All other authors report no disclosures.
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