Article

Excessive exercise training causes mitochondrial

functional impairment and decreases glucose
tolerance in healthy volunteers
Graphical abstract Authors
Mikael Flockhart, Lina C. Nilsson,
Senna Tais, Björn Ekblom,
William Apró, Filip J. Larsen

Correspondence
mikael.flockhart@gih.se (M.F.),
filip.larsen@gih.se (F.J.L.)

In brief
Flockhart and colleagues investigated the
dose-response relationship of exercise
training and found that excessive training
causes mitochondrial impairment and
reduced glucose tolerance in healthy
volunteers. Mitochondrial ROS
production was reduced, thereby
maintaining total oxidative stress. In a
separate cohort, disturbed glucose
control was found in free-living elite
endurance athletes.

Highlights
d Here, we investigate the dose-response effects of exercise
training in healthy subjects

d Excessive exercise training induces substantial

mitochondrial respiratory impairment

d Mitochondrial impairment is associated with impaired

glucose tolerance

d Despite excessive training, markers of global oxidative stress

were unchanged

Flockhart et al., 2021, Cell Metabolism 33, 957–970

May 4, 2021 ª 2021 Elsevier Inc.
https://doi.org/10.1016/j.cmet.2021.02.017 ll
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Article
Excessive exercise training causes mitochondrial
functional impairment and decreases
glucose tolerance in healthy volunteers
Mikael Flockhart,1,* Lina C. Nilsson,1 Senna Tais,1 Björn Ekblom,1 William Apró,1,2 and Filip J. Larsen1,3,*
1The Swedish School of Sport and Health Sciences, GIH, Åstrand Laboratory, Department of Physiology, Nutrition and Biomechanics,
Stockholm 114 33, Sweden
2Department of Clinical Sciences, Intervention and Technology, Karolinska Institutet, Stockholm, Sweden
3Lead contact

*Correspondence: mikael.flockhart@gih.se (M.F.), filip.larsen@gih.se (F.J.L.)

https://doi.org/10.1016/j.cmet.2021.02.017

SUMMARY

Exercise training positively affects metabolic health through increased mitochondrial oxidative capacity and
improved glucose regulation and is the first line of treatment in several metabolic diseases. However, the up-
per limit of the amount of exercise associated with beneficial therapeutic effects has not been clearly iden-
tified. Here, we used a training model with a progressively increasing exercise load during an intervention
over 4 weeks. We closely followed changes in glucose tolerance, mitochondrial function and dynamics, phys-
ical exercise capacity, and whole-body metabolism. Following the week with the highest exercise load, we
found a striking reduction in intrinsic mitochondrial function that coincided with a disturbance in glucose
tolerance and insulin secretion. We also assessed continuous blood glucose profiles in world-class endur-
ance athletes and found that they had impaired glucose control compared with a matched control group.

INTRODUCTION The quality of the mitochondrial population and its effects on

Mitochondria are the primary sources of cellular ATP synthesis
and are major ROS (reactive oxygen species)-producing organ-
elles and are thus central in energy and redox metabolism. The
size and function of the mitochondrial pool are vital for metabolic
health and muscular function. Mitochondrial capacity is tightly
correlated to whole-body maximal oxygen uptake (van der
Zwaard et al., 2016), which itself is a strong proxy for metabolic
function and health (Lee et al., 2011). A reduced mitochondrial
function is often (Bhatti et al., 2017; Kelley et al., 2002; Lowell
and Shulman, 2005; Simoneau et al., 1999), but not always
(Boushel et al., 2007; Lefort et al., 2010), observed in insulin-
resistant subjects. Studies using transgenic rodents with severe
reductions in mitochondrial content have a paradoxically
improved glucose tolerance (Zechner et al., 2010). A mismatch
between fatty acid availability and mitochondrial fat oxidation
has been implicated in the accumulation of ectopic fat and cer-
amide, which is a common finding in insulin-resistant skeletal
muscles (Pan et al., 1997). Therefore, it is highly debated whether
mitochondrial dysfunction itself is a cause or a consequence of
impaired blood glucose control and insulin sensitivity. The term
‘‘mitochondrial dysfunction’’ has no clear definition, and mito-
chondrial efficiency and ROS production (Konopka et al.,
2015), intrinsic mitochondria respiration (Mogensen et al.,
2007), and mitochondrial density (Morino et al., 2005) have all
been reported to differ between insulin-resistant subjects and
controls.
metabolic health is not merely regulated by the mitochondrial
number and its respiratory capacity. An emerging field of
research has shown that the location and function of the mito-
chondria within the cell is highly dynamic and regulated by fusion
and fission events, creating a large communicative mitochon-
drial network (Westermann, 2010). In many pathological states,
mitochondria display a reduced ability to respond to nutrient
supply and match mitochondrial respiration to metabolic de-
mand (Nunnari and Suomalainen, 2012). In addition, the tran-
scription factor NF-E2 p45-related factor 2 (Nrf2) and the E3
ligase adaptor Kelch-like ECH-associated protein 1 (KEAP1),
which is the primary negative regulator of Nrf2, are both critical
players in redox homeostasis, the antioxidative response, and
mitochondrial biogenesis. It was recently shown that acute exer-
cise increases the total Nrf2 protein content within minutes after
an exercise bout (Gallego-Selles et al., 2020), but less is known
regarding the chronic response to training.
Exercise training has proven to be a powerful tool to stimulate
mitochondrial biogenesis and can act as a preventative treat-
ment against many metabolic disorders by stimulating glucose
uptake (Pedersen and Saltin, 2015). However, an upper limit
where the exercise stimuli no longer result in further positive
metabolic outcomes has not been clearly identified. The benefits
of exercise training on cardiac health seem to follow a curvilinear
relationship where modest to high investments in physical activ-
ity appear to be most beneficial. High to extreme amounts of ex-
ercise training have been associated with negative effects on

Cell Metabolism 33, 957–970, May 4, 2021 ª 2021 Elsevier Inc. 957
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cardiac health that include increased coronary artery calcifica-
tion, myocardial fibrosis, and arrhythmia. Although a reversed
J-shaped association between the amount of exercise and
health outcomes have been demonstrated, no clear threshold
between optimal and excessive amounts of exercise has been
established (Eijsvogels et al., 2018; Franklin et al., 2020).
In competitive sports, it is well established that an appropriate
exercise stimulus improves performance, whereas too much
training leads to staleness and reduced performance. In the
short-term perspective, this state is termed as nonfunctional
overreaching, which can develop into overtraining syndrome in
the long term (Halson and Jeukendrup, 2004). In a study in which
12 subjects performed sprint interval training, we found a sub-
stantial reduction in mitochondrial respiration with oxidative al-
terations of mitochondrial proteins (Larsen et al., 2016). As the
mitochondrial function is tightly associated with metabolic
health, such derangements should have negative metabolic out-
comes. Therefore, we hypothesized that there is a bell-shaped
relationship between exercise training load and mitochondrial
function, glucose metabolism, and physiological adaptation to
exercise training in human subjects, during a training program
with a progressive increase in training load.
RESULTS
To test our hypothesis, we recruited six female and five male
healthy subjects to take part in a 4-week training intervention
consisting of high-intensity interval training (HIIT; see Table S1
for subject characteristics). During the first 3 weeks, we carefully
titrated the exercise sessions more and more often and were
thereby able to study the physiological responses to different
loads of exercise training. During the fourth week, the exercise
load was reduced to allow for recovery. Throughout the interven-
tion, muscle biopsies and oral glucose tolerance tests (OGTTs)
were performed to assess the metabolic response in different
phases. See Figure 1A for schematics of the study design, Fig-
ure 1B for exercise training load, and Figure S1 for the time
points of biopsy and OGTTs. A detailed description of the
method is presented in the STAR methods section.
Performance and cardiovascular adaptations to
progressive HIIT
The HIIT used during the study was proven effective in enhancing
performance and physiological capacities. Power output during
HIIT increased from baseline (BL) through light training (LT) and
moderate training (MT) (Figures 1C and 1D). In the phase of
excessive training (ET), despite increasing the training load, the
improvement in physical performance stagnated, indicating
what others have described as accumulating fatigue and malad-
aptation to the exercise stimuli (Halson and Jeukendrup, 2004).
After the recovery phase (RE), when the training load was dras-
tically reduced, performance peaked (Figure 1C). In contrast to
the dynamic response in physical performance, maximal oxygen
consumption increased consistently throughout the study,
regardless of the training phase (Figure 1E). Maximal attainable
heart rate (Figure 1F) during HIIT training was suppressed after
ET but was restored after the RE, and heart rate response to sub-
maximal work followed a similar pattern (Figure 1G).
958 Cell Metabolism 33, 957–970, May 4, 2021
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With regard to adaptations on the whole-body level, our data
show the expected physiological changes after a period with
progressively harder training, commonly referred to as ‘‘over-
reaching,’’ and after a week with drastically reduced training,
the performance was increased in line with the concept of super-
compensation (Issurin, 2010).
Mitochondrial respiration is reduced after excessive
training with a compensatory increase in markers of
mitochondrial density
The classic cellular response to exercise training is an increase in
the amount and respiratory capacity of mitochondria that in-
creases the respiratory capacity of a given unit of muscle (Hol-
loszy, 1967). Intrinsic mitochondrial respiration (IMR) is instead
the respiratory capacity related to a mitochondrial parameter,
often a mitochondrial protein or citrate synthase (CS) activity.
We have previously shown an impaired IMR after intense
short-term sprint interval training (Larsen et al., 2016), and scat-
tered reports of impaired IMR have been found after extreme
endurance events (Konopka et al., 2017) as well as acutely after
a 5-km cycling time trial (Layec et al., 2018). Knowing that phys-
ical performance and VO2max increased in an expected manner
during the training period, we wanted to determine how these
changes were reflected at the mitochondrial level. We, therefore,
measured ex vivo IMR in isolated mitochondria from vastus later-
alis, to determine how this parameter responded to the different
training phases in this study.
During the first part of the intervention, from BL to LT and MT,
IMR numerically increased (from 13.2 ± 1.8 to 15.7 ± 1.4
pmol$s 1$mg 1) but decreased drastically by 40% (to 9.4 ± 1.1
pmol$s 1$mg 1) during ET compared with MT (Figure 2A). In
RE, intrinsic mitochondrial function seemed to recover numeri-
cally but was still 25% lower than that during MT (11.8 ± 1.1
pmol$s 1$mg 1) (Figure 2A). Compared with MT, the reduction
in mitochondrial intrinsic respiration after ET was evident,
regardless of the substrate combination used, which decreased
by 45% (from 8.3 ± 0.9 to 4.6 ± 0.5 pmol$s 1$mg 1) when
respiring on complex I substrates (Figure 2B) and decreased
by 42% (from 9.0 ± 0.9 to 5.2 ± 0.4 pmol$s 1$mg 1) in the pres-
ence of succinate and rotenone that support complex II respira-
tion (Figure 2C). Similar reductions in IMR were seen when
respiration was normalized to CS activity in the mitochondrial
suspension (Figure S2). However, in LEAK state (in the absence
of ADP but in the presence of respiratory substrates pyruvate,
glutamate, and malate) (Figure 2D) as well as for ADP-stimulated
respiration on the lipid substrate octanoyl carnitine and malate,
no decreases were found in IMR after ET (Figure 2E). The respi-
ratory control index was 17.3 ± 1.1 on an average and was unaf-
fected throughout the intervention (Figure 2F), and the externally
added cytochrome c had no significant effect on respiration (Fig-
ure 2G). This indicates a high quality of our mitochondrial fraction
that remained consistent during the intervention.
The amount of protein and activity of the mitochondrial
enzyme CS is often used as a quantitative estimate of the mito-
chondrial content and serves as a stable marker for long-term
adaptations to the endurance exercise (Vigelsø et al., 2014). Us-
ing a spectrophotometric approach, we measured CS activity in
muscle homogenate and observed a steady increase throughout
the training period (Figure 2H). To further assess the effect of
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Figure 1. Schematics of study design and performance and cardiovascular adaptations to progressive HIIT
(A) During the intervention, 14 sessions of HIIT training were performed on a cycle ergometer, of which 6 were combined with power output and physiological
measurements of VO2, CO2, blood lactate, blood glucose, and heart rate. HIIT sessions started with a standardized warm-up of 10 min at 100 W at 70 rpm,
followed by five 4-min-long intervals at ~95% of VO2max with 3 min of rest. The HIIT sessions were scheduled in order to capture mitochondrial status and
physiological outcomes at BL, after an initial LT load, after MT load, after ET load, and after a period of RE in training load. During the training intervention, five
biopsies from vastus lateralis were taken and four OGTTs were conducted. Each biopsy and OGTT was performed in fasted state 14 h post exercise.
(B) The density of HIIT in the different phases expressed as minutes of HIIT per week.
(C and D) Cycling performance measured as mean power output during HIIT sessions (C) and change in power output session to session (D).
(E) Mean oxygen uptake during HIIT sessions.
(F) Maximal heart rate during HIIT sessions.
(G) Heart rate at a fixed submaximal intensity of 100 W.
Unless otherwise stated, a repeated-measure one-way ANOVA was used, and a significant main effect (of time) is marked with a # in graphs. Dunnett’s post hoc
test was used on normally distributed data and Dunn’s post hoc test was used on non-normal distributed data. Post hoc tests were performed between ET and all
other situations. All values are mean ± SEM. For all measurements, n = 11.

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Figure 2. Mitochondrial respiration decreases after excessive training with a compensatory increase in markers of mitochondrial density
(A) Maximal ADP-stimulated respiration for complex I+II in the presence of the respiratory substrates pyruvate, glutamate, succinate, and malate.
(B) Respiration through complex I using pyruvate, glutamate, and malate.
(C) Respiration through complex II using succinate as a respiratory substrate in the presence of rotenone to inhibit complex I.
(D) LEAK respiration (in the presence of pyruvate, glutamate, and malate but without added ADP).
(E) Fat oxidation stimulated by octanoyl carnitine, malate, and ADP.
(F) The respiratory control indices were calculated as complex I+II respiration divided by leak respiration.
(G) Addition of cytochrome c to assess membrane integrity in the mitochondrial fraction.
(H) The activity of citrate synthase in muscle homogenate was assessed spectrophotometrically.
(I–L) The abundance of CS protein (I), the mitochondrial outer membrane channel protein VDAC1 (J), and inner membrane organizer Mitofilin (K) were all measured
by the western blot technique. A reference memcode staining is presented in (L). For all measurements, n = 11.

exercise training on the abundance of mitochondrial proteins, we
also performed western blot analysis of CS protein, the voltage-
dependent anion-selective channel 1 (VDAC1) located in the
outer mitochondrial membrane, mitochondrial inner membrane
organizer Mitofilin, and proteins in the electron transport chain.
The abundance of CS protein displayed a numerical but nonsig-
nificant increase during the intervention (Figure 2I), whereas
VDAC1, Mitofilin, and complex I–IV increased throughout the
intervention (Figures 2J, 2K, and S3). Conclusively, mitochon-
drial content and enzymatic activity increased stepwise in an ex-
pected manner and stands in sharp contrast with the observed
decrease in IMR after the ET phase.
Excessive training reduces glucose tolerance while
maintaining metabolic flexibility during a glucose load
The mitochondrial dysfunction that is observed in T2D patients is
an intensely debated potential cause of insulin resistance in skel-
etal muscle (Pinti et al., 2019). If the mitochondrial function is an
important determinant of insulin sensitivity, the dramatic impair-
ment in IMR observed in this study could translate into a

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Figure 3. Changes in glucose tolerance, insulin secretion, GLUT4 abundance, and metabolic flexibility following progressive exercise
training
(A and B) Glucose tolerance was tested by administering a 75 g glucose solution in water after an overnight fast. Glucose was measured in venous blood samples
and the glucose area under the curve was calculated during the following 120 min.
(C) Correlation analysis of the change in mitochondrial respiration and the change in AUC for glucose between the different situations. The baseline values are
represented by the dot in origin. Group mean values in each situation are presented in the graph.
(D) Insulin was measured using a colorimetric ELISA assay.
(E and F) HOMA-IR (E) and HOMA-b (F) was calculated from glucose and insulin values at each time point.
(G) C-peptide was measured at each time point during the OGTT.
(H and I) GLUT4 protein in whole-muscle homogenate (H) and in a membrane-bound fraction (I) was measured using western blot. Memcode staining was used as
a loading control.

(legend continued on next page)

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disturbance in glucose control. Indeed, the 40% reduction in
IMR observed in ET surpasses the difference of ~20% in IMR
between T2D patients and healthy weight-matched controls pre-
viously reported (Mogensen et al., 2007). To elucidate if the
reduction in IMR in this study was associated with a disturbed
metabolic function, we performed repeated OGTT in combina-
tion with indirect calorimetry at four different time points (BL,
LT, ET, and RE) throughout the training intervention. We found
that glucose area under the curve (AUC) was unaltered during
the first part of the intervention (from 600 ± 29 at BL to 589 ±
22 mmol$L 1$min after LT) but were strikingly increased to
658 ± 23 mmol$L 1$min during ET (Figures 3A and 3B). Interest-
ingly, during the RE phase, glucose tolerance only partly recov-
ered, closely resembling the dysfunction in mitochondrial
respiration that showed the same pattern. In a sub-analysis,
we found that the change in glucose tolerance was significantly
correlated to the change in mitochondrial respiration, time point
to time point (Figure 3C).
The AUC for plasma insulin changed during the intervention
and decreased from 6,141 ± 890 after LT to 4,950 ±
556 mU$mL 1$min after ET (Figure 3D). Therefore, the calculated
HOMA-IR values were unchanged (Figure 3E) but HOMA-b
values were attenuated after ET and then recovered after RE
(Figure 3F). In contrast, C-peptide was unchanged throughout
the intervention (Figure 3G), indicating a higher hepatic clear-
ance of insulin in the ET phase.
Exercise training affects glucose uptake partly by increasing
the abundance of the glucose transporter 4 protein (GLUT4),
which is located in the cytosol during rest and is transported to
the membrane upon insulin stimulation (Richter and Hargreaves,
2013). We therefore measured the abundance of GLUT4 in mus-
cle biopsy samples collected before glucose intake and found
that, contrary to the impaired glucose uptake in ET, GLUT4 in
whole muscle increased and was most abundant after ET (Fig-
ure 3H). We also measured the fractional abundance of mem-
brane-bound GLUT4 but no differences were found between
the intervention phases (Figure 3I). The phosphorylation of Akt
substrate of 160 kDa (AS160) has been implicated in the translo-
cation of GLUT4 to the cell membrane upon insulin stimulation
(Mı̂inea et al., 2005). In agreement with the data on GLUT4 trans-
location, we did not find any changes in the total amounts of phos-
phorylated levels of AS160 in our intervention (Figures 3J and 3K).
The observed dysregulation of glucose control during ET
might be a consequence of reduced glucose uptake and/or by
a reduced capacity to oxidize glucose. The resting metabolic
rate (RMR) before glucose load was not different between
training situations (Figure S4), and the thermogenic effect of
the glucose load increased the metabolic rate by 8% but was
not different in any of the training situations. However, fat and
carbohydrate oxidation varied throughout the intervention with
the highest measurements made after LT and ET (Figure S5).
The change in substrate oxidation can, however, not explain
the observed shift in glucose tolerance between LT and ET as
substrate oxidation was unaltered between those time points.
(J and K) Total AS160 (J) and phosphorylated AS160 (K) were measured by the western blot technique.
(L) Fat oxidation during exercise at submaximal intensity (100 W) was calculated using indirect calorimetry.
(M) Pictures of protein staining for purity of fractions and a reference memcode staining.
For insulin, HOMA-IR and HOMA-b, n = 10; GLUT4 membrane fraction, n = 8. All other measurements, n = 11.
962 Cell Metabolism 33, 957–970, May 4, 2021
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Insulin resistance and type 2 diabetes (T2D) have been re-
ported to be associated with a reduced capacity to oxidize lipid
substrates in some studies (Kelley and Simoneau, 1994; Men-
sink et al., 2001). Others report no difference between type 2 di-
abetics and healthy subjects (Mogensen et al., 2007), whereas
some have found increased lipid oxidation and accumulation
of incomplete beta-oxidation metabolites in the insulin-resistant
state (Koves et al., 2008). Since the capacity to oxidize lipids is
not maximally stimulated during resting conditions, we wanted
to measure this capacity at a submaximal work rate (100 W).
This work rate corresponded to 48% ± 2% of the subjects
VO2max and is close to the relative work rate that elicits the high-
est rates of lipid oxidation (Jeukendrup and Wallis, 2005). We
found that the rate of lipid oxidation increased continuously
from BL (5.6 ± 0.9 kJ$min 1) and was the highest after ET
(11.0 ± 0.9 kJ$min 1), despite the substantial reduction in IMR
at this time point (Figure 3L). These results indicate that the ca-
pacity to oxidize lipids is not responsible for the reduced glucose
tolerance in this setting. The subjects remained weight stable
during the intervention (69.4 ± 11.2 kg at BL and 69.0 ±
11.0 kg at ET; Figure S6) and, therefore, we rule out the possibil-
ity that an energy deficit could explain the observed metabolic
dysregulation.
Excessive training attenuates the glucoregulatory
response to intense exercise despite increased skeletal
muscle glycogen stores
High-intensity exercise is enabled through a parallel activation of
both oxidative and glycolytic energy pathways. Interestingly,
lactate has recently been shown to be a primary circulating
fuel that can be used as a carbon source in the tricarboxylic
acid cycle and thereby constitutes a route for the distribution
of mitochondrial fuel across the circulation (Hui et al., 2017).
Therefore, we analyzed blood glucose and lactate during exer-
cise throughout the intervention.
During submaximal work, glucose was not significantly
affected (Figure 4A), and during HIIT, both blood glucose and
lactate increased as expected compared with submaximal in-
tensity. This increase was, however, sharply attenuated as
training load increased and was lowest after ET (Figures 4B
and 4C). The glycolytic energy contribution, therefore, appeared
inhibited after ET and is probably the main cause of the staled
cycling performance in ET. After RE, the metabolic switch back
to a more glycolytic mode during HIIT was remarkable with the
highest measurements of blood glucose and lactate as perfor-
mance peaked during HIIT.
The primary source of lactate originating from glycolysis is
skeletal muscle glycogen. To determine whether the blunted
lactate response to HIIT was an effect of the reduced availability
of glycogen in the skeletal muscles, we analyzed glycogen con-
centrations in muscle tissue from vastus lateralis. Surprisingly,
we found that glycogen was only reduced between BL and LT,
which probably is explained by the glycogen usage during the
first HIIT session and the limited time for repletion. Thereafter,
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Figure 4. Excessive training attenuates the glucoregulatory response to intense exercise despite increased skeletal muscle glycogen stores
(A) Blood glucose was measured after a submaximal work rate.
(B and C) Blood glucose (B) and lactate (C) after the last HIIT interval.
(D) Muscle glycogen content was measured in muscle homogenate from vastus lateralis using a spectrophotometric approach
(E) The activity of hexokinase was measured spectrophotometrically throughout the training period.
(F) Glycogen synthase was measured by the western blot technique.
(G) Fasting plasma free fatty acids were measured spectrophotometrically throughout the training period.
(H) A reference memcode staining.
For all measurements, n = 11.

glycogen increased throughout the different training phases (Fig-
ure 4D). Further, we analyzed two key proteins involved in the
synthesis of glycogen from glucose. The maximal activity of
hexokinase, which catalyzes the conversion of glucose to
glucose-6-phosphate, was increased after ET (Figure 4E) in
agreement with the higher glycogen depots at that time point.
Likewise, the amount of glycogen synthase protein increased af-
ter ET (Figure 4F). Thus, the reduced blood lactate concentration
measured in ET cannot be explained by reduced glycogen avail-
ability, which previously has been suggested to be linked to
staled performance during intensified training (Costill et al.,
1988). This indicates that other metabolic changes likely must
be important in the regulation of substrate use and performance
during intense training.
According to the Randle effect (Randle et al., 1963), a stimu-
lated lipid metabolism through elevated levels of blood FFAs
should potentially inhibit glycolytic activity. As elevated circu-
lating FFAs also are associated with insulin resistance we
measured blood FFA after overnight fasting in our subjects
throughout the training period. However, the concentrations of
FFA were low and remained unaltered throughout the training
period, and thus, cannot explain the deterioration in glucose
tolerance or the decreased plasma lactate levels during HIIT in
the ET phase (Figure 4G).
Overall, ET appears to inhibit cycling performance through a
blunted glycolytic response to intense exercise, despite normal
muscle glycogen levels and unaltered metabolic stimulation
through plasma FFA supply.
Mitochondrial fusion and fission respond to exercise
training in a dose-dependent manner but are not
specifically modulated after excessive exercise
After moderate exercise training, mitochondrial dynamics have
been shown to be activated by the promotion of fusion and
reduction of fission events (Memme et al., 2021). The importance
of a functional mitochondrial reticulum is gaining increasing
attention as an important factor in explaining the health out-
comes of exercise training (Tanaka et al., 2020). For example,
mitochondrial fragmentation and fission have been implicated
in insulin resistance in skeletal muscle cells (Jheng et al.,

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Figure 5. Mitochondrial fusion and fission respond to exercise training in a dose-dependent manner but are not specifically modulated dur-
ing excessive exercise
The abundance of mitochondrial outer membrane fuser MFN2 (A), inner membrane fuser OPA1 (B), fission executor DRP1 (C), docking protein MFF (D), and FIS1
(E) all assessed using the western blot technique. Pictures of a reference memcode staining are presented in (F). For all measurements, n = 11.
2012), whereas overexpression of the fusion factors MFN 1 and
MFN 2 has an insulin-sensitizing effect (Lin et al., 2018).
We found that Mitofusin 2 (MFN2) (Figure 5A), which fuses the
mitochondrial outer membrane, as well as inner membrane fuser
optic atrophy 1 (OPA1) (Figure 5B) both increased robustly dur-
ing the first 3 weeks of progressive training load and were most
abundant after ET. The activation of mitochondrial fission did not
follow a uniform pattern and the protein content of DRP1 was not
significantly altered throughout the training period (Figure 5C). In
contrast, the abundance of the DRP1 receptor MFF decreased
during the intervention (Figure 5D), whereas FIS1 increased
and was highest after ET (Figure 5E).
In human skeletal muscle, the activation of autophagy seems
to be dependent on exercise intensity, duration, as well as en-
ergy availability (Martin-Rincon et al., 2018). We assessed three
canonical markers of autophagy. The microtubule-associated
protein 1 light chain (LC3BI), which creates docking sites for au-
tophagolysosomes and lysosomes, and the lipidated form
LC3BII, were unchanged throughout the training intervention
(Figures S7A and S7B). Beclin1, an important protein in the auto-
phagosome formation, increased during the intervention and
reached a peak value during ET (Figure S7C).
Taken together, the presumed scheme of healthy remodeling
and activation of autophagy appears to be activated in parallel
with increasing exercise volume and cannot explain the dramatic
reduction in IMR during the ET phase.
Oxidative stress is maintained during excessive training
owing to reduced mitochondrial H2O2 emission
ROS increase during exercise (Henrı́quez-Olguin et al., 2019) but
are effectively quenched by the antioxidant system that includes
mitochondrial superoxide dismutase (SOD2), catalase (CAT),
glutathione peroxidase (GPX), and hemeoxygenase (HO-1-1) un-
964 Cell Metabolism 33, 957–970, May 4, 2021
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der normal circumstances. Despite the increase in training load,
we found no significant effect on any of the antioxidative proteins
SOD2, GPX, CAT, and HO-1-1, which all remained unchanged
with training as assessed by western blotting (Figures 6A–6D).
To understand how increasing doses of exercise affected the
overall oxidation status in the skeletal muscles, we measured the
amount of peroxidized lipids and carbonylated proteins in mus-
cle homogenates taken throughout the study. We expected the
level of oxidative stress to increase with increasing exercise
training load. Surprisingly, we found that the total amount of per-
oxidized lipids (Figure 6E) and carbonylated proteins (Figure 6F)
were kept unchanged during the intervention.
ROS produced during exercise has been shown to originate
from xanthine oxidoreductase, NADPH oxidase, and the mito-
chondria themselves (Henrı́quez-Olguin et al., 2019). Knowing
that the overall oxidation status was maintained despite
increasing training loads, we wanted to test if mitochondrial
hydrogen peroxide production was altered in different training
phases.
We found that the production of H2O2 closely followed
changes in mitochondrial respiration and was reduced in ET
compared with the highest values in LT and MT. H2O2 production
was lower when both mitochondria respired on complex I sub-
strates alone (Figure 6G) and in a combination of complex I
and II substrates (Figure 6H). This decrease in mitochondrial
H2O2 production could be a compensatory mechanism to coun-
teract increases in non-mitochondrial ROS production as a pro-
tective strategy against oxidative stress.
We have previously reported the redox-sensitive TCA cycle
enzyme, aconitase, to decrease by over 50% in parallel with
mitochondrial respiration after sprint training (Larsen et al.,
2016). Here, we found that the mitochondrial aconitase activity
changed during the intervention with a small increase from BL
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Figure 6. Oxidative stress is maintained during excessive training owing to reduced mitochondrial H2O2 emission with a simultaneous loss
of Nrf2
(A–D) The abundance of proteins in the mitochondrial antioxidative defense system; SOD2 (A), GPX (B), CAT (C), and HO-1-1 (D) were measured using the western
blot technique.
(E) The amount of peroxidized lipids was measured with a TBARS assay.
(F) Carbonylated proteins were measured using a colorimetric approach.
(G and H) Mitochondrial H2O2 production while mitochondria respired on complex I substrates (G) and on both complex I+II substrates (H) were measured using
Amplex UltraRed and horseradish peroxidase in the high-resolution respirometer.
(I) Activity of TCA cycle enzyme aconitase was measured in isolated mitochondria using a colorimetric approach.
(J–L) The abundance of Nrf2 protein (J) and the abundance of Nrf2 retainer protein Keap1 (K) was measured using the western blot technique. The ratio between
Nrf2 and Keap1 is presented in (L).
(M) A reference memcode staining.
A repeated-measure one-way ANOVA was used and a significant main effect (of time) is marked with a # in graphs. Dunnett’s post hoc test was used on normally
distributed data and Dunn’s post hoc test was used on non-normal distributed data. (G) and (H) were assessed using a mixed-model analysis due to missing data.
Post hoc tests were performed between ET and all other situations with the exception of (J)–(L), where an uncorrected Dunn’s test was performed between ET and
MT (annotated with $ in J and K). (I) n = 10; for all other measurements, n = 11.

Cell Metabolism 33, 957–970, May 4, 2021 965

 ll
to MT and was followed by a reduction after ET (Figure 6I). The
decreased mitochondrial respiration could be interpreted as a
protective strategy to maintain redox homeostasis.
Mitochondrial functional impairment and decreased
glucose tolerance coincide with loss of Nrf2
Having established that mitochondrial respiration and H2O2
emission decreased after the ET phase, we wanted to find a
mediator of this effect. Nrf2 is the master regulator of several
hundred genes involved in the antioxidant defense but knock-
down of Nrf2 has also been shown to reduce mitochondrial
respiration and cellular ATP levels (Holmström et al., 2013). Like-
wise, Nrf2 seems to be dysregulated in diabetic patients (Rab-
bani et al., 2019), and mice lacking Nrf2 protein have disturbed
glucose homeostasis with insulin resistance after a period of ex-
ercise (Cox et al., 2018). Nrf2 abundance showed a rather high
intra-individual variability when the training load was low (in BL
and RE), and thus, no significant main effect of time was found
(p = 0.16, ANOVA). However, Nrf2 abundance decreased be-
tween the MT and ET phases, and an uncorrected Dunn’s test
between these two situations in isolation showed a significant ef-
fect (Figure 6J; p < 0.05). Similarly, the repressor KEAP1 reached
its highest value after ET (Figure 6K; ANOVA main effect, p =
0.12; uncorrected Dunn’s test, p < 0.05, ET compared with
MT). Therefore, the Nrf2/KEAP-1 ratio reached its lowest value
966 Cell Metabolism 33, 957–970, May 4, 2021
Article
Figure 7. Elite endurance athletes have
disturbed glucose control
Elite endurance athletes and a matched control
group used CGMs in periods up to 2 weeks.
(A–C) The number of minutes per 24-h period that
blood glucose was above 8 mmol$L 1 (A) between
4 and 8 mmol$L 1 (B) and below 4 mmol$L 1 (C).
(D) Mean glucose for athletes and controls over 24
h. Statistical analysis was done using an unpaired
Student’s t test. Athletes, n = 15; controls, n = 12.
after ET (Figure 6L; ANOVA main effect,
p = 0.24; uncorrected Dunn’s test, p =
0.08, MT compared with ET). Nrf2 protein
is continually synthesized and retained in
the cytosol by KEAP1; a decreased Nrf2/
KEAP1 ratio indicates less amounts of
protein that can translocate to the nucleus
where it binds to promoter regions of its
target genes. However, we found no
detectable amounts of Nrf2 in the nucleus
at any time point, possibly reflecting the
timing of the biopsy 14 h after the last ex-
ercise session.
Elite endurance athletes have a
disturbed glucose control
Knowing that ET has detrimental effects
on glucose tolerance and deranges the
glycolytic response to exercise, we
wanted to know if this was a problem in
a real-life setting. To test if the impair-
ments in glucose tolerance found in this study were mirrored
as an impaired glucose control in elite athletes, we recruited
a second cohort of 15 healthy endurance athletes representing
national teams in endurance sports and measured their glucose
control every 15th min for up to 2 weeks using continuous
glucose monitors (CGMs). Their results were compared with
age, sex, and a weight-matched control group of 12 subjects
that trained less than 7 h per week. For subject characteristics,
see Table S2.
We found that in free-living conditions, where athletes and con-
trols maintained their normal training and diet, the mean 24-h
glucose was nearly identical in athletes and controls (5.5 ± 0.1
versus 5.5 ± 0.1 mmol$L 1). Surprisingly, the time spent outside
of the normoglycemic range (4–8 mmol$L 1) was significantly
higher in athletes compared with controls. Athletes spent 41 min
per 24 h in the hyperglycemic range (>8 mmol$L 1) compared
with 22 min for the controls (Figure 7A) (t test, p = 0.005),
1,284 min in the normoglycemic range (4–8 mmol$L 1) compared
with 1,384 min for the controls (Figure 7B) (t test, p = 0.001), and
115 min in the hypoglycemic range (<4 mmol$L 1) compared
with 34 min for the controls (Figure 7C) (t test, p = 0.04). In the ath-
letes, hyperglycemia seemed to occur in the early afternoon and
hypoglycemia mainly occurred during sleep between 3 and 7
a.m. (Figure 7D). Interestingly, normoglycemia seemed to be
maintained in most cases even during extensive training sessions.

Article
DISCUSSION
Here, we show that there is an upper limit of the amount of inten-
sive exercise that can be performed without disrupting metabolic
homeostasis. Beyond this limit, negative effects on metabolic
health and adaptation of physical performance, seemingly
caused by a mitochondrial partial shutdown of both respiration
and H2O2 production, start to manifest. The results of this study
also provide valuable insight into the ongoing controversy about
whether mitochondrial dysfunction is a cause or occurs second-
ary to insulin resistance (Goodpaster, 2013; Holloszy, 2013). The
decreased IMR and glucose intolerance coincided with an unex-
pected loss of Nrf2 protein.
The exercise protocol used here with progressively
increasing training loads is a unique model allowing us to
examine the relationship between mitochondrial dysfunction,
metabolic health, and glucose intolerance in the same healthy
subjects over a short period of time. We found that changes
in IMR were closely paralleled by changes in glucose tolerance
(Figure 3C). These results support the hypothesis that mito-
chondrial dysfunction indeed causes disturbances in glucose
metabolism. The classical explanation for such impairment is
that dysfunctional mitochondria have a lower capacity to
oxidize lipid-based substrates that leads to pathological lipid
accumulation, which, in turn, is intimately associated with insu-
lin resistance (Morino et al., 2006). In this study, such an expla-
nation is unlikely, primarily due to the fact that fat oxidation at a
submaximal work rate (100 W) increased throughout the
training phases, regardless of the drastic impairment in IMR,
including mitochondrial fat oxidation, in the ET phase. The
rate of fat oxidation during the submaximal work rate was 3.7
times higher than at rest, indicating that the mitochondrial ca-
pacity for fat oxidation should be far more than the demand
during resting conditions. In conjugate, these results suggest
a dissociation between whole-body fat oxidation and the
maximum mitochondrial capacity for fat oxidation and suggest
that other factors contribute to the metabolic disturbances
observed in ET.
The interplay between IMR and insulin sensitivity is complex.
In a study comparing healthy controls with offspring of patients
with T2D, exercise training increased mitochondrial ATP produc-
tion in both groups but only improved insulin sensitivity in the
controls (Irving et al., 2011). In another study, obese insulin-
resistant women had similar IMR but higher mitochondrial
H2O2 emission than a lean control group. After 12 weeks of
training, mitochondrial H2O2 emission decreased in the obese
group in parallel with an increased insulin sensitivity, whereas
these parameters were unchanged in the control group (Ko-
nopka et al., 2015).
The reduction in IMR in the ET phase contrasts the response in
markers of mitochondrial density. CS enzyme activity, as well as
mitochondrial protein abundance, including VDAC1 and Mitofi-
lin, all peaked during ET. This indicates that the ET phase
activated mitochondrial biogenesis pathways and more mito-
chondrial proteins were synthesized, but they had a lower IMR.
Therefore, the mitochondrial dysfunction observed in this study
does not seem to involve a disturbance in the biogenesis of mito-
chondria or a decreased mitochondrial number, as shown in
some early studies on insulin resistance (Kelley et al., 2002),
ll
but is rather an intrinsic mitochondrial defect, as shown in pa-
tients with T2D (Mogensen et al., 2007).
We hypothesized that during excessive exercise the ROS pro-
duction should increase and overwhelm the endogenous antiox-
idative systems. Surprisingly, we found that both the number of
carbonylated proteins and TBARS were kept unchanged
throughout the study, indicating that the total level of ROS pro-
duction was constant. In the ET phase, mitochondrial H2O2 pro-
duction was reduced by more than 50% compared with MT. We
interpret this finding as a compensatory partial shutdown of
mitochondrial metabolism to reduce overall ROS production
and maintain a balanced redox environment. The reduction in
IMR can only partly be explained by the inactivation of the
redox-sensitive aconitase. When mitochondrial respiration was
assessed with the complex II substrate succinate together with
the complex I inhibitor rotenone, aconitase was bypassed but
the magnitude of reduction in IMR in the ET phase was only partly
attenuated compared with complex I respiration. Inhibition of
mitochondrial respiration despite an increase in mitochondrial
proteins has previously been found in a model with transient
knockdown of SOD2 during embryonic development, where it
was interpreted as an adaptive response to oxidative stress
(Cox et al., 2018).
The training load during the ET phase consisted of almost daily
intervals at the all-out effort and must be considered a rather
extreme type of exercise training that only highly motivated indi-
viduals can tolerate for more than a few days in a row. Therefore,
we do not see a high risk that people wishing to improve their
health through exercise enter the state of mitochondrial impair-
ment and glucose intolerance due to ET volumes. However, our
finding that the cohort of elite endurance athletes has a disturbed
glucose control compared with healthy controls is in line with the
hypothesis that ET can cause glucose intolerance. This finding is
highly surprising, knowing that athletes with high oxidative capac-
ity are assumed to have high insulin sensitivity, and during aging,
they show less decline in metabolic health than controls (Borgh-
outs and Keizer, 2000). To the best of our knowledge, only one
study has assessed continuous glucose profiles in athletes,
wherein ~40% of the subjects were found to spend a substantial
amount of time in the hyperglycemic range (Thomas et al., 2016).
The negative metabolic effects of excessive exercise found in this
study are likely reduced as soon as the training load decreases.
Indeed, we found a substantial improvement in metabolic param-
eters after only 1 week of reduced training in the RE compared
with the ET phase.
From a health perspective, we do not advise against intensive
exercise training as former elite athletes have lower mortality
rates and seem to live longer compared with the general popula-
tion (Ruiz et al., 2014). Nevertheless, both athletes and those
looking to improve their health through exercise should carefully
monitor the response to training, as too much exercise might
have negative effects. Using changes in glucose tolerance or
careful tracking of glucose homeostasis using CGM could be a
minimally invasive and a novel approach to optimize the amount
of exercise associated with the greatest benefits.
Limitations of study
We used healthy, active subjects and do not know how seden-
tary subjects or patients with metabolic disease respond to
Cell Metabolism 33, 957–970, May 4, 2021 967
 ll
Article

increasing doses of HIIT exercise. In this cohort, 90 min of HIIT
per week was well tolerated, whereas 152 min per week was
associated with maladaptations. The amount of training associ-
ated with optimal adaptations is likely lower in subjects not
accustomed to exercise training, whereas elite athletes should
tolerate a higher training load after years of training. Another lim-
itation is the timing of the biopsies and venous blood samples,
which were taken ~14 h after the last training session. It is
possible that IMR, mitochondrial H2O2 emission, and blood
FFA showed an acute response to each exercise session with
more reliance on fat oxidation and more severe impairments of
IMR immediately after each exercise session and that these pa-
rameters had partly recovered by the time point of our biopsy
collection.
STAR+METHODS
AUTHOR CONTRIBUTIONS
M.F., L.C.N., S.T., W.A., and F.J.L. performed experiments. M.F. and L.C.N.
supervised training sessions. M.F., L.C.N., W.A., and F.J.L. analyzed data.
W.A. and B.E. performed muscle biopsies and B.E. had the medical responsi-
bility. F.J.L. conceived the hypothesis, designed the study, and finalized the
manuscript. M.F. wrote the first draft and finalized the manuscript. All authors
have read, commented on, and approved the final version of the manuscript.
DECLARATION OF INTERESTS
F.J.L. is co-founder of Silicon Valley Exercise Analytics, a company using data
science to improve athletic performance. The company had no role in funding,
data collection and analysis, or preparation of the manuscript. All other authors
declare no conflict of interest.
Received: July 12, 2020
Revised: November 18, 2020
Accepted: February 22, 2021
Published: March 18, 2021

Detailed methods are provided in the online version of this paper

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STAR+METHODS

KEY RESOURCES TABLE

REAGENT or RESOURCE SOURCE IDENTIFIER

Antibodies
Anti-Heme Oxygenase 1 antibody (HO-1-1) Abcam Cat#ab13248; RRID: AB_2118663
Anti-Glutathione Peroxidase 1 Abcam Cat#ab22604; RRID: AB_2112120
antibody (GPX)
Anti-Catalase antibody - Peroxisome Abcam Cat#ab16731; RRID: AB_302482
Marker (CAT)
Anti-SOD2/MnSOD antibody (SOD2) Abcam Cat#ab13533; RRID: AB_300434
Anti-Mitofusin 2 antibody [6A8] (MFN2) Abcam Cat#ab56889; RRID: AB_2142629
Anti-OPA1 antibody [EPR11057(B)] (OPA1) Abcam Cat#ab157457; RRID: AB_2864313
Anti-DRP1 antibody [EPR19274] (DRP1) Abcam Cat#ab184247
Anti-TTC11/FIS1 antibody [EPR8412] (FIS1) Abcam Cat#ab156865
Anti-MFF antibody [EPR7360] (MFF) Abcam Cat#ab129075; RRID: AB_11155454
Anti-VDAC1 / Porin antibody Abcam Cat#ab154856; RRID: AB_2687466
[EPR10852(B)] (VDAC1)
Anti-Mitofilin antibody [EPR8749] (Mitofilin) Abcam Cat#ab137057
Anti-Glucose Transporter GLUT4 Abcam Cat#ab654; RRID: AB_305554
antibody (GLUT4)
Anti-Keap1 antibody (Keap1) Abcam Cat#ab139729
Anti-Citrate synthetase antibody Abcam Cat#ab129095; RRID: AB_11143209
[EPR8067] (CS)
LONP1 polyclonal antibody Abnova Cat#PAB28578
Beclin-1 (D40C5) Rabbit mAb (Beclin1) Cell Signaling technology Cat#3495; RRID: AB_1903911
LC3B (D11) XP Rabbit mAb (LC3B) Cell Signaling technology Cat#3868; RRID: AB_2137707
Glut4 (1F8) Mouse mAb (GLUT4) Cell Signaling technology Cat#2213; RRID: AB_823508
NRF2 (D1Z9C) XP Rabbit mAb (Nrf2) Cell Signaling technology Cat#12721; RRID: AB_2715528
p70 S6 Kinase Antibody (p70) Cell Signaling technology Cat#9202; RRID: AB_331676
eEF2 Antibody (eEF2) Cell Signaling technology Cat #2332; RRID: AB_10693546
Glycogen Synthase Antibody (GS) Cell Signaling technology Cat #3893; RRID: AB_2279563
AS160 Antibody (AS160) Cell Signaling technology Cat #2447; RRID: AB_2199376
Phospho-AS160 (Thr642) (D27E6) (pAS160) Cell Signaling technology Cat#8881; RRID: AB_2651042
Anti-mouse IgG, HRP-linked Antibody Cell Signaling technology Cat#7076; RRID: AB_330924
Anti-rabbit IgG, HRP-linked Antibody Cell Signaling technology Cat#7074; RRID: AB_2099233
Chemicals, peptides, and recombinant proteins
SuperSignal West Femto Maximum Thermo Scientific Cat#34096
Sensitivity Substrate
Pierce Bovine Serum Albumin Standard Thermo Scientific Cat#23208
Pre-Diluted Set
Pierce(R) 660nm Protein Assay Reagent Thermo Scientific Cat#22660
Restore PLUS Western Blot Stripping Thermo Scientific Cat#464030
Buffer
Pierce Reversible Protein Stain Kit for PVDF Thermo Scientific Cat#24585
Membranes
Critical commercial assays
Subcellular Protein Fractionation Kit for Thermo Scientific Cat#87790
Tissues
C-peptide ELISA Mercodia Cat#10-1136-01; RRID: AB_2750847
Insulin Human ELISA Kit Invitrogen Cat# KAQ1251
(Continued on next page)

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Continued
REAGENT or RESOURCE SOURCE IDENTIFIER
Lipid Peroxidation (MDA) Assay Kit Sigma-Aldrich Cat#MAK085
Aconitase Assay Kit Abcam Cat#ab83459
Protein Carbonyl Content Assay Kit Abcam Cat#126287
Software and algorithms
DatLab 5.2 software Oroboros https://www.bioblast.at/index.php/
MitoPedia:_DatLab
ChemiDoc XRS system with Quantity One Bio-Rad https://www.bio-rad.com/en-se/product/
software (version 4.6.3) quantity-one-1-d-analysis-software?
ID=1de9eb3a-1eb5-4edb-82d2-
68b91bf360fb
ChemiDoc MP with Quantity One software Bio-Rad https://www.bio-rad.com/en-se/product/
(version 6.0.1) quantity-one-1-d-analysis-software?
ID=1de9eb3a-1eb5-4edb-82d2-
68b91bf360fb
Graph Pad prism 8.3.1 for Windows Graph Pad https://www.graphpad.com/support/faq/
prism-831-release-notes/

RESOURCE AVAILABILITY

Lead contact
Further information and requests for resources and reagents as well as datasets and protocols should be directed to and will be ful-
filled by the Lead Contact, Filip J Larsen (filip.larsen@gih.se).

Materials availability
This study did not generate new unique reagents.

Data and code availability

This study did not generate/analyze datasets/code.

EXPERIMENTAL MODEL AND SUBJECT DETAILS

Subjects
We recruited six female and five males to take part in the intervention. All subjects that started the training intervention completed the
full protocol. They were all healthy and recreational active and conducted endurance and strength training on regular basis. Exclusion
criteria were commitment to systematic endurance training of more than 5 hours a week as well as regular high intensity interval
training (HIIT). Detailed subject characteristics are presented in Table S1. Female and male subjects were not divided into, or treated
as sub-groups during the intervention or in the reported results. Also, due to the time-span of the intervention and the unknown inter-
ference of hormonal status during the female subject’s menstrual cycle, they started the intervention at different time-points in their
menstrual cycle. Statistical analyses of our primary outcomes, mitochondrial respiration and glucose tolerance, did not reveal any
sex related differences.
As an observational part of the study, a separate cohort of elite endurance athletes and controls were involved for measurement of
continues blood glucose. The elite endurance athletes were competitive at national team level in endurance sports and the group of
weight, age and height matched controls were recreational active but not competitive athletes. The subjects were included in the two
groups based on their training history and involvement in endurance competition. Subject characteristics are presented in Table S2.
All subjects gave their informed consent prior to inclusion in the study. The study protocol was approved in December 2017 by the
regional ethics committee in Stockholm and conformed to the declaration of Helsinki. The first subject was recruited in February 2018
and the training intervention lasted until December 2019 for the last subject. The interventional study was registered as a clinical trial
in February 2021 and was given the clinical trial identifier NCT04753021.

Pre-test
For inclusion and assessment of baseline physiological characteristics during cycling, a pre-test was performed on a SRM ergometer
(Schoberer Rad Messtechnik, SRM, Ju €lich, Germany). For assessment of the physiological response to submaximal work rate a stan-
dard protocol at the institution was used which comprised of a series of five minutes long intervals separated with one minutes of rest
were capillary blood lactate and glucose were taken and analyzed using a Biosen C-Line Clinic (EKF-diagnostics, Barleben, Ger-
many). The work rate was set individually at 80-100 W at the first stage and thereafter increased with 15-30 W per stage until a sub-

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stantial blood lactate accumulation was evident. After a short rest, an incremental maximal exercise test was initiated to determine
VO2max. The test started at the work rate of the previous stage, and increased with 20-30 W $ min-1 until fatigue. VO2max was ex-
pressed as the average of the four consecutive highest 10-second long periods, and Wmax as the power output reached before fa-
tigue. Breath by breath sampling of gas exchange were performed during the entire session using an Oxycon Pro device (Erich Jaeger
GmbH, Hoechberg, Germany). Heart rate was measured continuously (Polar Electro OY, Kempele, Finland) and the subjects rated
their perceived exertion using the BORG scale (Borg, 1982) at every stage and at exhaustion. The equipment was calibrated accord-
ing to manufactures instructions.

Training intervention
All physiological tests and HIIT sessions were carefully supervised by highly experienced personnel with extensive background in
physiological and performance testing. Subjects were blinded to their performance during HIIT sessions throughout the intervention
and were instructed to perform all sessions with the ambition to produce the highest possible mean power output. The intervention
period consisted of 14 HIIT-sessions in total all performed at the laboratory under close surveillance and careful monitoring of power
output and heart rate. In addition during six HIIT-sessions, respiratory gas exchange and capillary lactate were monitored with mea-
surements of performance and physiological response to exercise (referred to as HIIT-sessions in the article). The other 8 sessions
are referred to as HIITtraining sessions. The subjects were blinded to power output, cadence and heart rate during all HIIT and HIIT-
training
sessions. During HIIT sessions, VO2, CO2, blood glucose, lactate and rating of perceived exhaustion (BORG) were measured.
All HIIT sessions started with a submaximal warm up of 10 minutes at 100 W and 70 rpm. Substrate metabolism were calculated from
VCO2 and VO2 measurements using the Brouwer equation (Brouwer, 1957). Thereafter five 4 minutes long intervals followed, at
approximately 95% of VO2max intersected with 3 minutes of non-pedaling rest. The ergometer was set in constant brake mode
at an individually determined braking force. The subjects could then alter power output by variation in cadence or by choice of
gear. The subjects were instructed to perform all HIIT sessions to achieve the highest possible mean power output over all intervals.
In order to help the subjects with a recommended (standardized) pacing strategy they were paced at the first interval at a power
output corresponding to their previously highest measured mean power during a HIIT session. The HIITtraining-sessions comprised
of the standardized warm up of 10 min at 100 W and five 8 minutes long intervals at approximately 90% of VO2max intersected
with 3 minutes of non-pedaling rest during the first three weeks of the study. During the fourth week when training load was reduced,
the HIITtraining sessions were shortened to 3x8 minutes and 3x4 minutes (repetitions and minutes). The subjects often trained in pairs
and additional ergometers were used (Monark LT2, Monark Exercise AB, Vansbro, Sweden) besides the SRM during the HIITtraining
sessions. Outside of the training in the laboratory, subjects were only allowed to perform low intensity exercise and strength training
for the upper body. All individually performed training sessions were scheduled to not interfere with performance and physiological
measurements during HIIT sessions.

Nutritional intervention
Energy intake was standardized for each subject during the day of test sessions. Prior to the first HIIT-session, no exercise was
performed but was standardized in terms of rest (48 hour without training) and diet (24 hour). Subjects had their own standardized
breakfast, lunch and additional snacks, which were consumed three hours before reporting to the laboratory in late afternoon for HIIT-
sessions. The meals were individually chosen by the subjects and reflected their normal nutritional intake, and comprised of mix of
carbohydrates, protein and fat and was repeated prior to all biopsies and OGTT. After each HIIT and HIITtraining session a recovery
drink containing 1 g $ kg-1 bw of carbohydrates and 0.25 g $ kg-1 bw of protein was ingested by the subjects. After HIIT sessions,
subjects were supplied with an evening meal consisting of 74 g carbohydrates, 38 g protein and 26 g fat that was consumed two
hours post exercise. They thereafter remained fasted and reported to the laboratory in early morning for an oral glucose tolerance
test (OGTT) and donation of a muscle biopsy. Therefore, each subject had their own individual schedule for strict timing of training
sessions, biopsies, OGTT and diet in order to standardize the last 24 hour prior to each OGTT.

Continues glucose monitoring

In the separate cohort of elite endurance athletes, we continuously assessed blood glucose every 15th minute for up to two weeks
using a skin-mounted sensor (FreeStyle Libre, Abbot) that measure glucose in the interstitial fluid. The sensor was fitted laterally on
the subject’s deltoideus and automatically stored measurements for a maximum of eight hours before it had to be emptied using a
portable reader. If the sensor failed to measure, or if the subject did not export data from the sensor to the reader within eight hour
from the last export, the sensor overwrites previously stored data and leave a gap in the exported text fil. All text-files were manually
checked and abnormal values were excluded. The first 12 hours of measurements were also excluded from all subjects due to dif-
ferences in time before readings appeared stable. In total, 26 687 separate measurements are used for analysis and the mean effec-
tive wear-time was ten days and seven hours for each subject.

METHOD DETAILS

Muscle biopsies
All biopsies were collected in fasted state in early morning following 14 hour of rest after the last HIIT session. Biopsies were taken
from alternating leg in randomized order between subjects from vastus lateralis. First, local anesthesia (2 % Carbocain, AstraZeneca,

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€lje, Sweden) was injected at the biopsy site. A small incision was made and approximately 150 mg of wet tissue was removed
Söderta
with a Weil-Blakesley chonchotome (Henriksson, 1979) or a 5 mm Bergström needle with manually applied suction (Evans et al.,
1982). A detailed description of the biopsy technique has previously been described (Ekblom, 2017). The muscle samples were
blotted and dissected clean from visual fat, connective tissue and blood and divided in to three portions; 50 mg to ice cold ISO-
medium for respirometrics and two portions of 50-100 mg to liquid nitrogen and thereafter stored in -80 C for later analysis.

Oral glucose tolerance test and resting metabolism

Each subject completed four OGTTs throughout the study. After arrival to the laboratory resting metabolic rate (RMR) was measured
in supine position by gas exchange measurement using mixing chamber method (Oxycon Pro) attached to a ventilated hood. There-
after a muscle biopsy was taken and a catheter was inserted into the antecubital vein of the forearm and 15 ml blood was drawn. After
10 minutes of passive rest 75 mg glucose dissolved in 300 ml water was ingested and at 0, 15, 30, 45, 60 75, 90, 105- and 120-minutes
after glucose intake a venous blood sample were taken and instantly analyzed for glucose with enzymatic method in the Biosen C-
Line Clinic. At time points 30, 60, 90- and 120-minutes a 15 ml sample of venous blood was taken and kept on ice. The sample were
centrifuged at 2800 rcf and the plasma was pipetted into eppendorf tubes and stored at -80 C until later analysis. Additional RMR
measurements were performed at 40 minutes after glucose ingestion to capture substrate oxidation at the time point when glucose
peaked as well as at the end of OGTT at 115 minutes. A schematic of time-points during OGTT is presented in Figure S1.

Mitochondrial isolation and respirometry

Mitochondria were isolated from 50 mg of muscle in isolation medium (Sucrose 100 mM, KCl 100 mM, Tris-HCl 50 mM, KH2PO4
1 mM, EGTA 100 mM, BSA 0.1%; pH 7.4 as described by Gnaiger and Kuznetsov, 2002). Isolation of mitochondria were performed as
previously described (Tonkonogi and Sahlin, 1997) with modifications (Larsen et al., 2011). Briefly, muscle was kept on ice and ho-
mogenized first by scissors and thereafter after addition of 0.2 mg ml-1 bacterial protease, further homogenization was performed in a
water-cooled glass homogenizer. The homogenate was centrifuged at 700 rcf at 4 C for 10 min in a 15 ml tube and the supernatant
was transferred to new 1.5 ml tubes and centrifuged at 10000 g at 4 C. The pellets were resolved and transferred to a single 1.5 ml
tube and centrifuged at 7000 rcf at 4 C for 5 min giving a resultant pellet that was liquated in 0.6 ml per initial mg wet weight of muscle
in preservation medium (EGTA 0.5 mM, MgCl2 6H2O 3 mM, K-lactobionate 60 mM, Taurine 20 mM, KH2PO4 10 mM, HEPES 20 mM,
Sucrose 110 mM, BSA 1 g L-1 Histidine 20 mM, Vitamin E succinate 20 mM, Glutathione 3 mM, Leupeptine 1 mM, Glutamate 2 mM,
Malate 2 mM, Mg-ATP 2 mM) (Gnaiger and Kuznetsov, 2002).
Mitochondrial respiration and H2O2 emission were measured using a two-channel high-resolution respirometer with an attached
fluorescent probe (Oxygraph-2k, Oroboros Instruments Corporation, Innsbruck, Austria). 10 ul of isolated mitocondria were added to
two 2 ml wells containing respiration medium MIR05 (EGTA 0.5 mM, MgCl2.6H2O 3 mM, K-lactobionate 60 mM, Taurine 20 mM,
KH2P04 10 mM, HEPES 20 mM, Sucrose 110 mM, BSA 1 g L-1). All experiments were performed at 37 C and O2 calibration and
H2O2 standard calibration was performed according to the manufactures’ instructions. The protocol for assessing respiration in
the different states were: Channel A: 1 U/mL Horseradish peroxidase + 10 mM amplex ultrared (H2O2 emission), octanoyl carnitine
0.2 mM + malate 0.5 mM (leak fat), 2.5 mM ADP (fat respiration), rotenone 0.5 mM + succinate 10 mM (Complex II), N2-induced
anoxia/reoxygenation, titration of FCCP 0.05 mM steps (uncoupled respiration). Channel B: 1 U/mL Horseradish peroxidase +
10 mM amplex ultrared (H2O2 emission), pyruvate 5 mM + glutamate 10 mM + malate0.5 mM (Leak PGM), ADP 0.75 mM (complex
I), succinate 10 mM (Complex I+II), N2-induced anoxia/reoxygenation and cytochrome C 10 mM (membrane integrity). Mitochondrial
respiration in all states are related to protein content analyzed in the mitochondrial suspension using spectrophotometric analysis
and Pierce 660 nm protein assay (Thermo Fisher Scientific). All measurements were performed and analyzed in DatLab 5.2 software
(Oroboros, Paar, Graz, Austria).

Immunoblotting
Homogenization was performed in freeze dried samples of 2 mg muscle. The protocol for homogenization is extensively described
earlier (Samuelsson et al., 2016). Briefly, 100 ml/mg dry WT of homogenization buffer consisting of 2 mM HEPES (pH 7.4), 1 mM EDTA,
5 mM EGTA, 10 mM MgCl2, 50 mM b-glycerophosphate, 1% Triton X-100, 1 mM Na3VO4, 2 mM dithiothreitol, 1% phosphatase in-
hibitor cocktail (Sigma P-2850) and 1% (vol/vol) Halt Protease Inhibitor Cocktail (Thermo Scientific, Rockford, IL) was added to each
sample and processed in a bullet blender until homogenized. After rotation for 30 min at 4 C and centrifugation at 10,000 g for 10 min
at 4 C the supernatant was analyzed for protein content and diluted with homogenization buffer and Laemmli buffer (Bio-Rad, Rich-
mond, CA) obtaining a protein concentration of 1 mg/ml. All samples were denatured at 95 C for five minutes and stored at -80 C.
For analyses of cellular fractions a commercial kit was used (Subcellular protein fractionation kit for tissues, Thermo Scientific
87790). 40 mg of wet weight muscle was used for homogenization and treated according to the protocol generating a cytoplasmic
fraction, a membrane fraction and a nuclear fraction. Each fraction was analyzed for protein content and diluted with homogenization
buffer and Laemmli buffer for immunoblotting obtaining a protein concentration of 0.3, 0.06 and 0.25 mg/ml, respectively.
Immunoblotting was performed by the method described extensively in Moberg et al. (2016). Briefly, samples of muscle homog-
enates (20, 16 or 14 mg of protein) were loaded onto 16 or 26-well Criterion TGX gradient gels (4–20% acrylamide; Bio-Rad). Due to
dilution of samples 4.2 mg were loaded of the cytosolic fraction, 0.84 mg of the membrane fraction and 3.5 mg of the nuclear fraction
onto 26-well gels. Electrophoresis (300 V for 32 minutes kept on ice) were performed in transfer buffer containing 25 mM Tris base,
192 mM glycine, and 10% methanol and the proteins were then transferred to a polyvinylidine fluoride membranes (Bio-Rad) at a

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constant current of 300 mA for 3 h kept on ice. The membranes were stained with MemCode Reversible Protein Stain Kit (Thermo
Scientific) to be used as a loading control. The membranes were then destaind and blocked with Tris-buffered saline (TBS; 20 mM
Tris base, 137 mM NaCl, pH 7.6) containing 5 % nonfat dry milk for 1 h at room temperature. Thereafter, incubation overnight followed
with antibodies diluted with TBS buffer with 2.5 % non-fat dry milk and 0.1% Tween. The antibodies used were from Abcam; HO-1-1
(ab13248), GPX (ab22604), CAT (ab16731), SOD2 (ab13533), MFN2 (ab56889), OPA1 (ab157457), DRP1 (ab184247), FIS1
(ab156865), MFF (ab129075), VDAC1/Porin (ab154856), Mitofilin (ab137057) GLUT4 (ab654), KEAP1 (ab139729) and CS
(ab129095), from Abnova; LONP1 (PAB28578) and from Cell Signaling Technology; BECLIN1 (3495), LC3B (3868s), GLUT4
(2213), Nrf2 (12721), p70 (9202), eEF2 (2332) GS (3893), AS160 (2447), pAS160 (8881). After incubation, the membranes were washed
and incubated with secondary antibodies conjugated with horseradish peroxidase (Cell Signaling Technology; anti-mouse 7076s,
anti-rabbit 7074s) for 1 h at room temperature. The membranes were washed again and Super Signal West Femto Chemiluminescent
Substrate (Thermo Scientific) was added and target proteins visualized and quantified in Molecular Imager ChemiDoc XRS system
with Quantity One software (version 4.6.3; Bio-Rad) and in ChemiDoc MP with Quantity One software (version 6.0.1; Bio-Rad). When
applicable, membranes were stripped with Restore Western Blot Stripping Buffer (Thermo Scientific) before block in TBS+milk and
incubation with new primary antibodies. All target proteins were loaded so that the proteins of each subject were treated and exposed
to the same conditions as well as visualization and quantification. All proteins from muscle samples are related to eEF2-protein
located at 90-100 kD on the memcode with exception to Nrf2, GLUT4 and Keap1, GS, AS160, eEF2 and p70 which are related to
whole lanes. The cytosolic, membrane and nucleic fractions are all related to whole lanes.

Free fatty acids

Plasma FFA concentration were analyzed enzymatically in blood plasma by letting FFA react with Acyl-CoA synthase and later form
the product NAD+. The formation of NAD+ was measured at 340 nm during 120 minutes in a spectrophotometer (TECAN Infinite 200
Pro) and the amount of FFA’s were calculated. Method description is found in Miles et al. (1983).

Insulin
Plasma levels of insulin were measured using an ELISA kit (Invitrogen, KAQ1251) according to the manufacturer’s instructions.

C-peptide
Plasma levels of C-peptide were measured using an ELISA kit (Mercodia, Uppsala, Sweden, Cat # 10-1136-01) according to the man-
ufacturer’s instructions.

Muscle glycogen
Muscle glycogen was analyzed in 2 mg of freeze dried and dissected muscle using a homogenization protocol described earlier
(Leighton et al., 1989) with small modifications. Briefly, the samples were heated in 1 M KOH (70 C for 20 min) and thereafter incu-
bated in NaAc+HAc and NaAc+amyloglucosidase (40 C for two hour). Reagents and samples were added to cuvettes and incubated
for 20 minutes before reading of absorbance at 340 nM in a spectrophotometer (Beckman Coulter DU800).

Hexokinase activity
The activity of hexokinase was analyzed spectrophotometrically in homogenate from freeze dried muscle. The method has previously
been described (Grossbard and Schimke, 1966). Absorbance was measured over 20 minutes at 340 nm using a spectrophotometer
(TECAN Infinite 200 Pro).

CS activity
Citrate synthase activity were measured in 1-2 mg of freeze dried muscle. Extraction buffer (100ml/mg) containing 50 mM Tris, 1 mM
EDTA, and 5 mM MgCl2, pH 8.2 were added to the samples which were homogenized using a bullet blender. The supernatant was
transferred to new tubes and centrifuged at 1400 rcf rpm for 30 seconds and the pellet was discarded. Cuvettes was loaded with
reaction buffer containing 250 ml 100 mM Tris-HCl, 0.4 mM DTNB, 25 ml 0.1 mM acetyl-CoA 25 ml 1% triton-X, 170 ml H2O and
5 ml sample. Reaction was started with addition of 25 ml 7 Mm oxaloacetate. Absorbance were measured using spectrophotometric
method (Beckman Coulter DU800) at 412 nm at 25  C. CS-activity was also measured in the suspension of isolated mitochondria with
the same reagents.

Carbonyl content
Carbonyl content were analyzed in samples from 1 mg freeze dried muscle dissolved in 1M KOH and heated for 15 minutes at 70  C
using Abcam Protein Carbonyl Content Assay Kit (ab126287). Briefly, DNPH and TCA were added to samples, centrifuged for 5 mi-
nutes at 16 000 rcf and the pellet were dissolved in aceton, put in a bullet blender for mixing and then centrifuged for 2 minutes at 16
000 rcf. The resulting pellet were dissolved in guarnidine solution and added to a 96 wells microplate included in the kit for reading of
absorbance at 360 nm in spectrophotometer (TECAN Infinite 200 Pro). Samples were then moved to new wells and protein content
was determined using Thermo Scientific Pierce Protein Assay Kit (22660) and measured at 660nm.

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TBARS
Malondialdehyde (MDA), were analyzed using Sigma Aldrich Lipid Peroxidation Assay Kit (MAK085). The assay is based on the re-
action between MDA and thiobarbituric acid (TBA) forming a colorimetric product proportional to the MDA content. Homogenate (see
CS-activity for homogenate content) containing 1 mg freeze dried muscle was added to MDA standard solution and BHT. TBA-so-
lution were added to samples and incubated at 95  C for 60 minutes before absorbance was measured at 532 nm in a Beckman
Coulter DU800 Spectrophotometer.

Aconitase activity
Aconitase activity were measured in the mitochondrial fraction using assay kit (ab83459, Abcam). Absorbance was measured at
450 nm in a spectrophotometer (TECAN Infinite 200 Pro).

QUANTIFICATION AND STATISTICAL ANALYSIS

Repeated measures one-way ANOVA was used to analyze which of the measured parameters were affected by the progressive
training (main time effect). Normality was assessed using a Shapiro-Wilk test. If a significant main effect was found, Dunnetts
post-hoc test was used that controls for multiple comparisons. In case of non-normally distributed data Friedmans’ test was
used instead of a one-way ANOVA and Dunn’s post-hoc test was used to compare different time points. Post-hoc tests were per-
formed between the ET-phase and all other conditions with exception of Nrf2 and Keap1 (Figure 5) where an uncorrected Dunn’s test
were performed between ET and MT only. Correlations were calculated using Pearson’s correlation coefficient. When only two
groups were compared (elite vs control, Figure 7) unpaired Student’s t tests were used. In case of missing data (Figures 6G and
6H only) a mixed model analysis was utilized. All statistical analyses were performed in Graph Pad prism 8.3.1 for Windows (Graph-
Pad Software, San Diego, California USA) and a p value of <0.05 was considered significant (marked with # for main effect ANOVA,
with * for a significant difference between ET and other situations). Results are expressed as mean ± standard error of the mean
(SEM). Graphical abstract was created using Biorender.

ADDITIONAL RESOURCES

This trial was registered at clinicaltrials.gov with a clinical trial registry number: NCT047653021. The study was performed at Swedish
School of Sport and Health Sciences between February 2018 – February 2021

e6 Cell Metabolism 33, 957–970.e1–e6, May 4, 2021

Cell Metabolism, Volume 33

Supplemental information

Excessive exercise training causes mitochondrial

functional impairment and decreases
glucose tolerance in healthy volunteers
Mikael Flockhart, Lina C. Nilsson, Senna Tais, Björn Ekblom, William Apró, and Filip J.
Larsen
SUPPLEMENTAL TABELS AND FIGURES

Age Weight Height Body fat VO2max Wmax

n= (year) (kg) (cm) (%) (ml · kg-1 · min-1) (W)
Female 6 25 ± 3 61 ± 3 165 ± 1 16.8 ± 0.8 46.7 ± 1.8 233 ± 8
Male 5 31 ± 3 79 ± 3 179 ± 2 12.5 ± 3.0 50.5 ± 1.4 303 ± 18
All 11 27 ± 2 69 ± 3 171 ± 3 14.9 ± 1.5 48.4 ± 1.3 265 ± 14

Supplemental table 1. Subject characteristics in the training intervention. Related to figure 1.

VO2max and Wmax values were measured during cycling at the graded exercise test to fatigue
performed before the intervention started. Values are presented as mean ± S.E.M.

Age Weight Height

n= (year) (kg) (cm)
Athletes Female 6 26 ± 2 57 ± 3 169 ± 2
Control Female 7 27 ± 2 59 ± 2 167 ± 2
Athletes Male 9 29 ± 1 72 ± 2 182 ± 3
Control Male 5 26 ± 2 71 ± 3 176 ± 1

Supplemental table 2. Subject characteristics of endurance athletes and controls from

continuous glucose measurements (CGM). Related to figure 7.

Figure S1. Schematic of OGTT showing timing of biopsies, blood sampling and RMR
measurements. Related to figure 1.
Figure S2. Intrinsic mitochondrial respiration expressed per CS-activity in the same
mitochondrial fraction. Related to figure 2. All values as means ± S.E.M, n = 11.

Figure S3. The abundance of proteins in the electron transport chain for complex I (A), IV
(B), II (C) III (D) and V (E). Pictures of representative protein staining and a reference
memcode staining is presented in (F). A repeated measures one-way ANOVA was used and a
significant main effect (of time) is marked with a # in graphs. Dunnetts’ Post-hoc test was
used on normal distributed data and Dunn’s post-hoc test was used on non-normal distributed
data. Post-hoc tests were performed between ET and all other situations. Related to figure 2.
All values are mean ± S.E.M. For all measurements; n = 11.
Figure S4. Resting Metabolic Rate was measured using indirect calorimetry and a ventilated
hood while the subjects were resting in quiet and thermoneutral conditions. Related to figure
3. All values as means ± S.E.M, n = 11.

Figure S5. The relative ratio of substrate oxidation was calculated from the ratio of exhaled
carbon dioxide and consumed oxygen. Related to figure 3. All values as means ± S.E.M, n =
11.

Figure S6. Body weight measured before every HIIT session remained unchanged during the
intervention. Related to figure 3. All values as means ± S.E.M, n = 11.
Figure S7. The abundance of autophagy proteins LC3BI (A), LC3BII (B) and Beclin1
together with a reference memcode staining (C) were all assessed using western blot
technique. A repeated measures one-way ANOVA was used and a significant main effect (of
time) is marked with a # in graphs. Dunnetts’ Post-hoc test was used on normal distributed
data and Dunn’s post-hoc test was used on non-normal distributed data. Post-hoc tests were
performed between ET and all other situation. Related to figure 5. All values are mean ±
S.E.M. For all measurements; n = 11.