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ORIGINAL RESEARCH
published: 21 January 2020
doi: 10.3389/fendo.2019.00880
Frontiers in Endocrinology | www.frontiersin.org 1January 2020 | Volume 10 | Article 880
Edited by:
Gordon L. Klein,
University of Texas Medical Branch at
Galveston, United States
Reviewed by:
Peter Ebeling,
Monash University, Australia
Hasmik Jasmine Samvelyan,
Edinburgh Napier University,
United Kingdom
Craig Sale,
Nottingham Trent University,
United Kingdom
Gustavo A. Nader,
Pennsylvania State University (PSU),
United States
*Correspondence:
Louise M. Burke
louise.burke@ausport.gov.au
†These authors have contributed
equally to this work
Specialty section:
This article was submitted to
Bone Research,
a section of the journal
Frontiers in Endocrinology
Received: 04 September 2019
Accepted: 02 December 2019
Published: 21 January 2020
Citation:
Heikura IA, Burke LM, Hawley JA,
Ross ML, Garvican-Lewis L,
Sharma AP, McKay AKA, Leckey JJ,
Welvaert M, McCall L and
Ackerman KE (2020) A Short-Term
Ketogenic Diet Impairs Markers of
Bone Health in Response to Exercise.
Front. Endocrinol. 10:880.
doi: 10.3389/fendo.2019.00880
A Short-Term Ketogenic Diet Impairs
Markers of Bone Health in Response
to Exercise
Ida A. Heikura 1,2† , Louise M. Burke 1,2
*†, John A. Hawley 2, Megan L. Ross 1,2 ,
Laura Garvican-Lewis 1,2 , Avish P. Sharma 1, 3, Alannah K. A. McKay 1, 4, Jill J. Leckey 2,
Marijke Welvaert 1,5,6 , Lauren McCall 7and Kathryn E. Ackerman 7,8
1Australian Institute of Sport, Canberra, ACT, Australia, 2Exercise and Nutrition Research Program, Mary MacKillop Institute
for Health Research, Australian Catholic University, Melbourne, VIC, Australia, 3Griffith Sports Physiology and Performance,
School of Allied Health Sciences, Griffith University, Gold Coast, QLD, Australia, 4School of Human Sciences (Exercise and
Sport Science), The University of Western Australia, Crawley, WA, Australia, 5University of Canberra Research Institute for
Sport and Exercise, Canberra, ACT, Australia, 6Statistical Consulting Unit, Australian National University, Canberra, ACT,
Australia, 7Division of Sports Medicine, Boston Children’s Hospital, Boston, MA, United States, 8Neuroendocrine Unit,
Massachusetts General Hospital and Harvard Medical School, Boston, MA, United States
Objectives: To investigate diet-exercise interactions related to bone markers in elite
endurance athletes after a 3.5-week ketogenic low-carbohydrate, high-fat (LCHF) diet
and subsequent restoration of carbohydrate (CHO) feeding.
Methods: World-class race walkers (25 male, 5 female) completed 3.5-weeks of
energy-matched (220 kJ·kg·d−1) high CHO (HCHO; 8.6 g·kg·d−1CHO, 2.1 g·kg·d−1
protein, 1.2 g·kg·d−1fat) or LCHF (0.5 g·kg·d−1CHO, 2.1 g·kg·d−1protein, 75–80%
of energy from fat) diet followed by acute CHO restoration. Serum markers of bone
breakdown (cross-linked C-terminal telopeptide of type I collagen, CTX), formation
(procollagen 1 N-terminal propeptide, P1NP) and metabolism (osteocalcin, OC) were
assessed at rest (fasting and 2 h post meal) and after exercise (0 and 3 h) at Baseline,
after the 3.5-week intervention (Adaptation) and after acute CHO feeding (Restoration).
Results: After Adaptation, LCHF increased fasting CTX concentrations above Baseline
(p=0.007, Cohen’s d=0.69), while P1NP (p<0.001, d=0.99) and OC (p<0.001,
d=1.39) levels decreased. Post-exercise, LCHF increased CTX concentrations above
Baseline (p=0.001, d=1.67) and above HCHO (p<0.001, d=0.62), while P1NP
(p<0.001, d=0.85) and OC concentrations decreased (p<0.001, d=0.99) during
exercise. Exercise-related area under curve (AUC) for CTX was increased by LCHF after
Adaptation (p=0.001, d=1.52), with decreases in P1NP (p<0.001, d=1.27)
and OC (p<0.001, d=2.0). CHO restoration recovered post-exercise CTX and CTX
exercise-related AUC, while concentrations and exercise-related AUC for P1NP and OC
remained suppressed for LCHF (p=1.000 compared to Adaptation).
Conclusion: Markers of bone modeling/remodeling were impaired after short-term
LCHF diet, and only a marker of resorption recovered after acute CHO restoration.
Long-term studies of the effects of LCHF on bone health are warranted.
Keywords: ketogenic diet, bone health, exercise, nutrition, endurance athletes
Heikura et al. Ketogenic Diet Impairs Bone Markers
INTRODUCTION
Despite the generally positive effects of exercise in promoting
bone health, bone injuries represent a challenge to consistent
training and competition in high performance sport (1). This, in
part, is due to the interaction of dietary factors (e.g., low energy
availability, poor vitamin D status, inadequate calcium intake)
with unique features of the exercise program [e.g., minimal
or excessive bone loading associated with weight- and non-
weight-bearing sports, poor biomechanics (1,2)]. Low energy
availability (a mismatch between energy intake and the energy
cost of exercise) occurs in both female and male athletes (2)
and impairs bone health via direct (uncoupled bone turnover
with increased resorption rates) and indirect (mediation by
reproductive and metabolic hormones) mechanisms (1). In
addition, carbohydrate (CHO) availability may also play a role
in bone health. Indeed, results from several studies show that
commencing endurance exercise with low compared to normal
or high glycogen availability stimulates the release of the cytokine
interleukin-6 (IL-6) from the exercising muscles (3,4). Among its
range of effects, IL-6 has been hypothesized to lead to enhanced
activity of the receptor activator of the nuclear factor K B-ligand,
which controls bone turnover by increasing osteoclastic activity
(thereby increasing bone breakdown) (5). In support of this
contention, bone resorption is acutely increased when CHO is
restricted before (6), during (7), and after (8) prolonged (1–2 h)
endurance (running) exercise, and may be linked to concomitant
increases in IL-6 concentrations (7). However, a recent study
has reported that acute reductions in CHO availability around
exercise mediated an increase in markers of bone resorption that
are independent of energy availability and circulating IL-6 (9).
Apparent effects on other markers of bone metabolism, such as
osteocalcin (OC) and the bone formation marker procollagen 1
N-terminal propeptide (P1NP) in these models have been small
(6–9), although a 24 h fast has been reported to reduce blood OC
concentrations in lightweight rowers (10).
Whether these changes in markers of bone metabolism persist
(or are amplified) after chronic exposure to low CHO availability
around exercise remains unknown, but is of relevance in view
of the promotion of a ketogenic low CHO-high fat (LCHF) diet
to athletes and its putative benefits for endurance performance
(11). To date, no studies have examined the effects of longer-
term restriction of CHO at rest or in relation to exercise, although
in animal models and children with intractable epilepsy, chronic
adaptation to a ketogenic LCHF diet is associated with poor bone
health (12–16). In view of our recent observations of increased
post-exercise IL-6 concentrations in elite race walkers following
a 3.5-week adaptation to a LCHF diet (17), we investigated
the interaction of this diet and strenuous exercise on markers
of bone modeling/remodeling as secondary outcomes of our
larger study.
METHODS
Participants
Thirty world-class athletes (25 male, 5 female race walkers; ages
27.7 ±3.4 yr, BMI 20.6 ±1.7 kg/m2) were recruited over
three separate training camps during preparation for the 2016
Summer Olympic Games and the 2017 World Championships,
and provided written informed consent in accordance with
the Human Ethics Committee of the Australian Institute of
Sport (ethics approval no. 20150802 and 20161201). Six male
participants undertook two camps, however two of these data
sets were incomplete due to insufficient tissue samples, resulting
in 4 participants who had completed two camps being included
in the final analysis. In addition, two additional (male) data sets
were excluded from the final analysis due to their inability to
complete one of the experimental trials due to injury (unrelated
to bone). Therefore, our final data set provided a total of 32
trials (n=28 participants, 23 males, 5 females) with data for
pre- (Baseline) and post-treatment (Adaptation), of which 18
trials (13 males, 5 females) also contributed to data from acute
restoration to a HCHO diet (Restoration). Participants and elite
coaches contributed to the concept and implementation of the
research camps, helping to prioritize the themes of interest
and contributing to the design of the training program and
test protocols.
Study Overview
Participants completed a 3.5-week block of intensified training
and laboratory and field testing, supported by either a high-
CHO (HCHO) or an isoenergetic LCHF diet (Figure 1,Table 1),
consumed under strict dietary control (18). Upon completion
of the 3.5-week dietary intervention, a subset of participants
(n=18) completed a further testing block under conditions
of acute high CHO availability. Markers of bone metabolism
were measured after an overnight fast, in response to an energy-
matched meal of nutrient composition matching the intervention
diet, and in response to a bout of strenuous exercise (19), at
Baseline, Adaptation, and Restoration (Figure 1).
Dietary Control
Details of dietary control are described briefly here; more details
are described in prior work (18). Participants were allocated
into HCHO and LCHF groups based on preference. Both diets
were isocaloric (Table 1), however dietary CHO and fat intakes
differed between groups during intervention. Study diets were
designed and individualized for each athlete by trained members
of the research team including registered sports dietitians, a
professional chef, and exercise physiologists. All meals were
weighed (food scales accurate to 2 g) and provided for athletes
at set meal times. In addition, a collection of snacks per
individual meal plans were provided to the athletes each day. Any
unconsumed items or changes made to menu plans were weighed
and recorded for final analysis of dietary intakes. Compliance
to the meal plans was assessed daily. Meal plans were designed
and final dietary analysis of actual intakes was conducted using
FoodWorks 8 Professional Program (Xyris Software Australia Pty
Ltd, Australia). Further analysis of intakes was completed using
Microsoft Excel.
Experimental Design
Testing at Baseline, Adaptation, and Restoration involved a
hybrid laboratory/field test of 25 km (males) or 19 km (females)
Frontiers in Endocrinology | www.frontiersin.org 2January 2020 | Volume 10 | Article 880
Heikura et al. Ketogenic Diet Impairs Bone Markers
FIGURE 1 | Study flowchart and overview. Thirty-two data sets were gathered from 30 participants who participated in one or more training camps. After Baseline
testing on a carbohydrate-rich (HCHO) diet, they elected to follow a 3.5-week energy-matched dietary intervention of either HCHO or ketogenic low
carbohydrate-high fat (LCHF) principles. After Adaptation, the participants underwent an acute period of Restoration of high carbohydrate availability. At Baseline and
at the end (Adaptation) of this intervention, as well as after acute carbohydrate reintroduction (Restoration) they undertook a test block including a 25km (2 h) hybrid
laboratory/field race walking protocol at ∼75% VO2max. Venous blood samples were collected after an overnight fast, 2 h after an energy-matched breakfast based
on their diet (immediately pre-exercise), immediately post exercise and after 3 h of passive recovery during which an intervention-matched recovery shake was
consumed at 30 min. Blood samples were analyzed for serum concentrations of C-terminal telopeptide of type I collagen (CTX), procollagen 1 N-terminal propeptide
(P1NP), and osteocalcin (OC).
Frontiers in Endocrinology | www.frontiersin.org 3January 2020 | Volume 10 | Article 880
Heikura et al. Ketogenic Diet Impairs Bone Markers
TABLE 1 | Dietary intakes in the HCHO and LCHF groups.
Intervention Restoration
HCHO (n=14) LCHF (n=18) HCHO (n=8) LCHF (n=10)
Energy (kJ·d−1) 14,518 ±2,142 15,138 ±2,104 13,705 ±1,948 15,706 ±1,774
Energy (kJ·kg·d−1) 229 ±13 227 ±23 219 ±16 239 ±27
Protein (g·d−1) 133 ±22 143 ±19 132 ±24 151 ±18
Protein (g·kg·d−1) 2.1 ±0.2 2.1 ±0.2 2.1 ±0.2 2.3 ±0.2
Fat (g·d−1) 74 ±14 318 ±45*** 77 ±14 95 ±12**$$$
Fat (g·kg·d−1) 1.2 ±0.1 4.8 ±0.5*** 1.2 ±0.1 1.4 ±0.2**$$$
CHO (g·d−1) 549 ±75 35 ±5*** 492 ±60$552 ±62$$$
CHO (g·kg·d−1) 8.7 ±0.4 0.5 ±0.1*** 7.9 ±0.6$$ 8.4 ±1.0$$$
HCHO, high carbohydrate diet; LCHF, low carbohydrate high fat diet; CHO, carbohydrate.
**p<0.01, ***p<0.001 significant difference between diets.
$p<0.05, $$p<0.01, $$$ p<0.001 significantly different compared to Intervention.
at around 50 km race pace (75% of maximal oxygen uptake [VO2
max]) (Figure 1). Upon entering the laboratory in an overnight
fasted and rested state between 0600 and 0800 in the morning
(times were kept consistent within-participant), a cannula was
inserted into an antecubital vein for collection of blood samples
at rest (Fasting), immediately before exercise (2 h post-meal),
immediately after exercise (Post-ex) and 3 h post-exercise (3 h
post-ex). Blood was analyzed for concentrations of cross-linked
C-terminal telopeptide of type I collagen (CTX), P1NP and
total OC to determine the effects of dietary interventions and
exercise on bone metabolism. The cannulas were flushed with
3 ml of saline every 30 min throughout the trials. A standardized
breakfast (2 g·kg−1CHO for both groups during Baseline
and Restoration, or an isocaloric low CHO option for LCHF
during Adaptation) was consumed 30 min after the first blood
sample, after which the participants rested for 120 min before
beginning the session. During the Baseline and Restoration
exercise test, both groups ingested glucose (60 g·h−1) throughout
the test, while during Adaptation, isocaloric high fat snacks
were provided for the LCHF group. Upon completion of the
exercise test, the participants rested in the laboratory for a further
3 h, and received a standardized recovery shake (1.5 g·kg−1
CHO for both groups during Baseline and Restoration, or an
isocaloric low CHO option for LCHF during Adaptation; both
shakes included 0.3 g·kg−1protein) at 30 min post-exercise to
improve satiety.
Analysis of Serum Bone
Modeling/Remodeling Biomarkers
Blood samples were collected into a 3.5 mL EDTA BD Vacutainer
Plus SST II tube, and allowed to clot by standing at room
temperature for 2 h before centrifuging at 1,000 G for 10 min
for subsequent analysis of serum markers of bone resorption
(CTX), bone formation (P1NP) and overall bone metabolism
(OC). Analysis was undertaken by chemiluminescence on IDS-
iSYS (Immunodiagnostic Systems Limited; Boldon, Tyne and
Wear, UK). Inter-assay coefficient of variation as reported by
the manufacturer was 6.2, 4.6, and 6.1%, respectively. CVs
were determined as follows: OC: 6 serum controls were run,
using 3 reagents lots, in duplicate twice per day for 20 days,
on 2 analyzers; P1NP: 3 serum controls were run, using 3
reagent lots, in quadruplicates once per day for 20 days, on
2 analyzers; CTX: 5 serum controls were run, using 3 reagent
lots, in duplicate twice per day for 20 days, on 3 analyzers. In
addition to these tests, the laboratory ran quality control samples
throughout testing and the results were within the established
acceptable manufacturer ranges. The raw data for the analyses
of serum bone modeling/remodeling markers are provided in the
Supplementary Table to this publication.
Statistical Analyses
Statistical analyses were conducted using SPSS Statistics 22
software (INM, New York, USA) and R (R Core Team, 2018)
with a significance level set at p≤0.05. Normality of data was
checked with a Shapiro-Wilk test and visual inspection of residual
plots. General Linear Mixed models were fitted using the R
package lme4 (20) and included random intercepts for Subjects
and Camps to account for baseline inter individual heterogeneity
and the partial cross-over design. Because the estimated Camp
effect variance was 0, this random intercept was subsequently
removed to resolve boundary issues in the Restricted Maximum
Likelihood estimation. P-values were obtained using Type II
Wald F tests with Kenward-Roger degrees of freedom. Initial
models included all possible interactions but non-significant
interaction terms were dropped for ease of interpretation. Fasting
values and exercise-related area under curve [AUC; Pre-exercise
to 3 h post-exercise (21)] for all markers were compared with
a two-way mixed analysis of variance (ANOVA), with post-
hoc tests of Student’s t-tests for independent samples (between-
groups) and for paired samples (within-groups); where normality
was violated, Wilcoxon’s test and Mann-Whitney U-test were
used. Where a data point was missing, AUC was not calculated;
this resulted in exclusion of 1 participant in the CTX AUC
calculations, and 2 participants from both P1NP and OC
calculations. Effect sizes were calculated based on the Classical
Cohen’s dwhile accounting for the study design by using the
square root of the sum of all the variance components (specified
random effects and residual error) in the denominator. Data are
presented as means (95% confidence intervals [CI]).
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Heikura et al. Ketogenic Diet Impairs Bone Markers
RESULTS
Bone Modeling/Remodeling Biomarkers
During Fasting
Compared to Baseline, fasting concentrations of CTX were
increased after the LCHF diet (+22% [9, 35]: p=0.008, d=0.69),
with a decrease in P1NP (−14% [−19, −9]; p=0.001, d=0.99)
and OC (−25% [−35, −14]; p<0.001, d=1.39) levels
(Figure 2). In addition, the change in fasting P1NP (p<0.001,
d=1.64) and OC (p<0.001, d=1.78) after the 3.5-
week intervention was significantly different between the diets
(Figure 2).
Exercise Bone Markers
CTX decreased post-meal independent of dietary intervention
(Figures 3A,4A,p<0.001, d=1.63). At Adaptation, post-
exercise CTX concentrations in LCHF increased above Baseline
(p=0.001, d=1.67) and HCHO (p<0.001, d=0.62)
(Figure 3A). LCHF decreased P1NP (Figure 3B,p<0.001,
d=0.85) and OC across exercise (Figure 3C,p<0.001,
d=0.99) compared to Baseline. At Restoration, post-exercise
CTX returned to Baseline levels for LCHF (Figure 4A,p>0.05,
d=0.20 compared to Baseline), while concentrations of P1NP
(Figure 4B,p<0.001, d=0.23) and OC (Figure 4C,p<0.001,
d=0.21) remained suppressed across exercise.
Bone Marker Exercise Area Under Curve
At Adaptation, LCHF exercise-related AUC for CTX was greater
[+81% (54, 109); p<0.001, d=1.52] than Baseline, and
higher than HCHO (p=0.035, d=0.81) (Figure 3D). Exercise-
related AUC for P1NP decreased at Adaptation for LCHF
[−19% (−25, −12); p=0.003, d=1.27] compared with
Baseline and was lower than HCHO (p=0.009, d=1.03)
FIGURE 2 | Percentage change in fasting serum C-terminal telopeptide of
type I collagen (CTX), procollagen 1 N-terminal propeptide (P1NP) and
osteocalcin (OC) for high carbohydrate (HCHO; solid bars) and low CHO high
fat (LCHF; striped bars) after the 3.5-week dietary intervention. Data are means
±standard deviations. ***p<0.001 Significant between-group difference;
##p<0.01; ### p<0.001 Significant change from Baseline within-group.
(Figure 3E), with similar outcomes for OC [−29% (−35, −23);
p<0.001, d=2.0 and p<0.001, d=1.64, Figure 3F]. At
Restoration, LCHF experienced a return of exercise-related AUC
for CTX back to Baseline values [−43% (−21, 31); p=0.003,
d=1.08 compared to Adaptation and no difference compared
to HCHO; Figure 4D], meanwhile AUC for P1NP [+3% (−17,
48), p=1.000 compared to Adaptation and p=0.009, d=1.50
compared to HCHO; Figure 4E], OC [−3% (−19, 14), p=1.000
compared to Adaptation and p=0.010, d=1.47 compared to
HCHO; Figure 4F] remained suppressed.
DISCUSSION
Our data reveal novel and robust evidence of acute and likely
negative effects on the bone modeling/remodeling process in
elite athletes after a short-term ketogenic LCHF diet, including
increased marker of resorption (at rest and post-exercise) and
decreased formation (at rest and across exercise), with only
partial recovery of these effects following acute restoration of
CHO availability. Long-term effects of such alterations remain
unknown, but may be detrimental to bone mineral density
(BMD) and bone strength, with major consequences to health
and performance. While ketogenic diets are of interest to athletes
due to their ability to induce substantial shifts in substrate
metabolism, increasing the contribution of fat-based fuels during
exercise (11), we have previously reported the downside of a
concomitantly greater oxygen cost and reduced performance of
sustained high-intensity endurance exercise (19). The current
study identifies further complexity in the interaction between
the ketogenic diet and exercise with respect to markers of
bone modeling/remodeling, in which catabolic processes are
augmented and anabolic processes are reduced.
The LCHF diet is also popular within the general community
for its purported health benefits, including rapid weight loss
and improved glycemic control (22). However, data from
animal studies (12,13) demonstrate that chronic LCHF diets
are associated with impaired bone growth, reduced bone
mineral content, compromised mechanical properties, and
slower fracture healing. Furthermore, increased bone loss has
been reported in children with intractable epilepsy placed on
a medically supervised LCHF diet for 6 months (14,15). In
contrast, adults with type 2 diabetes mellitus who self-selected
to consume a LCHF diet for 2 years experienced no changes in
spinal BMD in comparison to a “usual care” group (22). One
explanation for these divergent outcomes involves interactions
of the LCHF diet with the level of habitual contractile activity.
Indeed in mice, a LCHF diet negated the positive benefits of
exercise on BMD in trabecular bone (16), while in children
with epilepsy, the rate of bone loss was greater in the more
active patients (14). Therefore, the hormonal response to exercise
undertaken with low CHO availability was of particular interest
in our study.
Previous studies involving acute strategies of low CHO
availability around exercise have identified effects on bone
resorption, as measured by increased blood CTX concentrations.
For example, males who undertook 60 min of treadmill running
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Heikura et al. Ketogenic Diet Impairs Bone Markers
FIGURE 3 | Time course of changes in bone marker concentrations across exercise (left panel) and exercise area under curve (right panel) for serum C-terminal
telopeptide of type I collagen (CTX) (A,D), procollagen 1 N-terminal propeptide (P1NP) (B,E), and osteocalcin (OC) (C,F) after the 3.5-week dietary intervention. Black
bars/symbols represent Baseline, gray bars/symbols represent Adaptation. Squares and circles represent high carbohydrate (HCHO) and low carbohydrate high fat
(LCHF), respectively. Gray bars represent a hybrid laboratory/field 19–25 km walk test at ∼75% VO2max. Data are means ±standard deviations. ##p<0.01;
###p<0.001 denotes significant differences at time points or tests within diet groups. *p<0.05; **p<0.01; ***p<0.001 denotes significant differences between
diet groups at a specific time point.
at 65% VO2max following a CHO-rich breakfast (∼1 g·kg1)
showed small variations in CTX responses, but only around the
exercise period, while dietary effects on parathyroid hormone,
OC and P1NP were not detected (6). Meanwhile, a more
strenuous protocol (120 min at 70% VO2max) was associated
with an attenuation of acute (pre-exercise to 2 h post-exercise)
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Heikura et al. Ketogenic Diet Impairs Bone Markers
FIGURE 4 | Time course of changes in bone marker concentrations across exercise (left panel) and exercise area under curve (right panel) for serum C-terminal
telopeptide of type I collagen (CTX) (A,D), procollagen 1 N-terminal propeptide (P1NP) (B,E), and osteocalcin (OC) (C,F) after acute reintroduction of carbohydrate
(right panel). Gray bars/symbols represent Adaptation, and white bars/symbols represent Restoration. Squares and circles represent high carbohydrate (HCHO) and
low carbohydrate high fat (LCHF), respectively. Gray bars represent a hybrid laboratory/field 19–25 km walk test at ∼75% VO2max. Data are means ±standard
deviations. $$p<0.01; $$$ p>0.001 denotes significant within-group difference compared to Restoration. *p<0.05; **p<0.01; ***p<0.001 denotes significant
differences between diet groups at a specific time point.
concentrations of IL-6, CTX, and P1NP when CHO was
consumed (0.7 g·kg·h1) during exercise (7). However, OC was
unchanged by diet and no differences in markers of bone
metabolism were detected over the subsequent three days,
suggesting that these effects are transient and quickly reversed
(7). Short-term effects were also reported when 24 elite male
runners with energy-matched intake over an 8 d period were
divided into a group who consumed CHO before, during, and
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Heikura et al. Ketogenic Diet Impairs Bone Markers
immediately after each of their 13 training sessions (additional
total CHO) while the others consumed an artificially sweetened
placebo (23). Here, CTX concentrations were suppressed at
80 min of recovery following an interval training sessions in
the CHO group with no dietary effects on P1NP or OC;
furthermore, fasting concentrations of all markers were similar
at baseline and on the ninth morning (23). Finally, Hammond
and colleagues (9) investigated the independent effects of low
CHO availability and acute energy restriction during the recovery
from one session of high-intensity interval running and the
completion of a subsequent session (3.5 h into recovery). They
reported lower CTX concentrations in the high CHO (control)
diet compared with both of the other conditions across the
various acute responses to exercise-related feeding, while there
were no differences between the energy and CHO restricted trials.
Meanwhile, only energy restriction produced an increase in IL-
6 responses to exercise, and there were no differences in P1NP
concentrations between dietary treatments (9). Furthermore, 5
d of low vs. optimal energy availability, which also resulted in
a 2-fold difference in CHO availability, was shown to result in
a significant difference in the AUC of fasting CTX (+85 vs.
+15%, respectively) and P1NP (−60 vs. −25%, respectively)
(24). To date, the only study to report an effect of acute
manipulations of CHO around exercise on bone formation
markers was that of Townsend et al. (8), in which the immediate
consumption of a protein-CHO feeding after a run to exhaustion
at 75% VO2max was associated with a suppression of the post-
exercise rise in CTX levels and a higher concentration of P1NP.
These authors concluded that immediate post-exercise meal
ingestion may benefit bone health compared to delayed feeding,
although the effects on CTX concentrations were reversed at
4 h post-exercise and a similar time course of P1NP changes
was not provided; therefore, it appears that the overall effect
on bone modeling/remodeling processes appears to follow meal
ingestion patterns.
The novelty of the current study was the interrogation of
the effects of prolonged adaptation to CHO restriction on bone
metabolism. Unlike the previous investigations, we identified
clear and consistent effects on bone metabolism at rest and in
response to exercise following 3.5-weeks of a ketogenic LCHF
diet (Figures 2–4), with increases in a marker of bone resorption
(CTX) and decreases in markers of bone formation (P1NP) and
metabolism (OC). Although some might argue that a complete
adaptation to a LCHF diet requires much longer than the 3.5-
week period utilized in the current study, it should be noted that
adaptations in substrate metabolism and exercise economy have
been reported across this (19,25), and much shorter (26), time
periods. Nevertheless, the current study is reflective of a shorter-
term adaptation to a LCHF diet and our findings warrant further
investigation across longer time periods.
Acute restoration of high CHO availability was only partially
effective in reversing these outcomes. Here, marker of bone
resorption returned to baseline with high CHO pre-exercise meal
and CHO ingestion throughout exercise, while the other markers
of bone metabolism remained suppressed, indicating impaired
overall balance of bone metabolism. This supports the concept
proposed by Hammond et al. (9) that CTX is responsive to acute
intake of CHO, possibly mediated through enteric hormone
secretion. Meanwhile, differences in muscle glycogen content,
which are not addressed by studies of acute feedings, may have a
greater effect on OC and P1NP concentrations. Given the serious
nature of injury risks and long-term outcomes of poor bone
health in later life in endurance athletes, further consideration
of the potential effects of the LCHF diet in exacerbating existing
risk factors for poor bone health is warranted. In particular, we
note that the impairment of bone metabolism around exercise
and recovery would involve a significant portion of the day in
athletes who undertake multiple training sessions, as well as being
superimposed on the changes identified at rest.
The interaction of diet and exercise on bone metabolism is
complex and requires more sophisticated investigation including
replication of the current findings. Furthermore, evolving
knowledge of inter-organ crosstalk suggests that outcomes
of altered bone metabolism may be more far-reaching than
the fate of the structural integrity of bone. Indeed, we note
the recognition of muscle and bone as endocrine organs,
with evidence that IL-6 released from contracting muscle has
autocrine, paracrine and endocrine effects (27). This includes
a purported feed-forward loop in which contraction-induced
stimulation of osteocalcin in myofibers promotes the release of
IL-6 and enhances muscle adaptation to exercise (27). Results
of the current study challenge this synergistic relationship
between osteocalcin signaling and IL-6, and remind us of
the pleiotropic nature of the molecules stimulated by diet-
exercise interactions.
Limitations
The data analysis undertaken in this study was a secondary
outcome of our investigations of the ketogenic LCHF diet;
these were not specifically powered to optimally address the
potential effects on markers of bone modeling/remodeling.
However, the detection of changes in the IL-6 response to
prolonged exercise in our initial study (12) provided motivation
to examine possible downstream effects. Because an identical
protocol was undertaken in two separate studies of the LCHF
diet, we were able to pool data from these investigations to
double the sample size previously known to allow detection of
changes in metabolism and performance. Indeed, changes in
markers of bone metabolism in the response to the interaction
of exercise and the dietary treatments were clearly detected
with the pooled data, but were also identifiable in the case
of the smaller sample size of the carbohydrate restoration
arm of the current dataset. Therefore, we feel confident that
our data are robust and warrant further investigation of
this theme.
CONCLUSIONS
Despite recent interest in the potential benefits of LCHF diets on
endurance performance or metabolic adaptation, the long-term
health effects of this dietary intervention are largely unknown.
We are the first to show that a 3.5-week ketogenic LCHF diet
in elite endurance athletes has negative effects on the markers of
bone modeling/remodeling at rest and during a prolonged high
Frontiers in Endocrinology | www.frontiersin.org 8January 2020 | Volume 10 | Article 880
Heikura et al. Ketogenic Diet Impairs Bone Markers
intensity exercise session. We also show only partial recovery
of these adaptations with acute restoration of CHO availability.
Given the injury risks and long-term outcomes underpinned by
poor bone health in later life, in athletes as well as individuals who
undertake exercise for health benefits, additional investigations
of the ketogenic diet and its role in perturbing bone metabolism
are warranted.
DATA AVAILABILITY STATEMENT
The datasets analyzed for this study were harvested from 2 trials
registered at Australian New Zealand Clinical Trial Registry
(ACTRN12619001015134 and ACTRN12619000794101), found
at: http://www.ANZCTR.org.au/ACTRN12619001015134.aspx
and http://www.ANZCTR.org.au/ACTRN12619000794101.aspx.
ETHICS STATEMENT
The studies involving human participants were reviewed and
approved by Australian Institute of Sport Ethics Committee. The
patients/participants provided their written informed consent to
participate in this study.
AUTHOR CONTRIBUTIONS
Conception and design of the experiments was undertaken
by IH, LB, MR, LG-L, AS, AM, JL, MW, LM, and KA.
Collection, assembly, analysis, and interpretation of data was
undertaken by IH, LB, MR, LG-L, AS, AM, JL, MW, LM,
and KA. Manuscript was prepared by IH, LB, KA, and JH.
All authors approved the final version of the manuscript. IH
and LB had full access to all the data in the study and take
responsibility for the integrity of the data and the accuracy of the
data analysis.
FUNDING
This study was funded by a Program Grant from the
Australian Catholic University Research Funds to Professor LB
(ACURF, 2017000034).
ACKNOWLEDGMENTS
We thank our research colleagues and supporters of the
Supernova research series and acknowledge the commitment of
the elite race-walking community.
SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found
online at: https://www.frontiersin.org/articles/10.3389/fendo.
2019.00880/full#supplementary-material
Supplementary Table 1 | Individual data for the serum concentrations of bone
modeling/remodeling markers (CTX, P1NP and Osteocalcin) in response to
strenuous exercise (2 h race walking) and dietary interventions (high carbohydrate
and low carbohydrate high fat diets) in elite race walkers.
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Conflict of Interest: The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be construed as a
potential conflict of interest.
Copyright © 2020 Heikura, Burke, Hawley, Ross, Garvican-Lewis, Sharma, McKay,
Leckey, Welvaert, McCall and Ackerman. This is an open-access article distributed
under the terms of the Creative Commons Attribution License (CC BY). The use,
distribution or reproduction in other forums is permitted, provided the original
author(s) and the copyright owner(s) are credited and that the original publication
in this journal is cited, in accordance with accepted academic practice. No use,
distribution or reproduction is permitted which does not comply with these terms.
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