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Life Sciences 251 (2020) 117603

Contents lists available at ScienceDirect

Life Sciences
journal homepage: www.elsevier.com/locate/lifescie

Lifelong treadmill training improves muscle function detected by a modified T

grip strength test during aging in BALB/c mice
⁎,1
Yu Zhao , Fei Shen1, Mingkai Gong, Ling Jin, Xiangyu Ren, Kailin Liu, Jian Lu
⁎

School of Physical Education & Health, East China Normal University, Shanghai 200241, China

ARTICLE INFO ABSTRACT

Keywords: Aims: The objective of this study was to establish a more accurate analytical model and method for grip strength
Lifelong exercise test of mice and to investigate the beneficial effects of lifelong exercise training to prevent functional decline of
Training muscle during aging.
Grip strength test Main methods: Fifty randomly selected adult male BALB/c mice (24–56 week age) were used in grip strength
Sarcopenia
testing periodically with short periods (3 s). Such method was used to detect a modified grip strength test. Then
Myonuclei
sixty-four male BALB/c mice (6 week age) were assigned into groups Sedentary (n = 32), Exercise (n = 32,
lifelong treadmill training 3 days per week and 30 min per day). The muscle strength and morphology para-
meters were measured at the ages of 6, 12, 30 and 64 weeks, respectively.
Key findings: The grip strength (peak value and endurance) of sedentary group monotonically decreased in adult
BALB/c mice during aging, while that of exercise group remained a relatively high level even slightly increased
at aged period. Meanwhile, lifelong exercise training could slow down the loss of gastrocnemius muscle mass,
myofiber cross-sectional area, myonuclear number and myonuclear domain. The number of myofibers was re-
latively stable in adult mice.
Significance: Modified analytical method for grip strength testing, with improved accuracy and reliability, may
be an efficient substitute for conventional method in measuring the strength and endurance of mice and in-
vestigating the effect of lifelong exercise on muscle loss. Lifelong exercise training helps prevent muscle loss and
muscle defunctionalization while aging.

1. Introduction
It is well established that physical inactivity may result in non-
communicable chronic diseases and death in the world [1,2]. Exercise
can improve health as well as alleviate the harmful effects of aging-
related disorders such as metabolic syndrome, cardiovascular diseases,
sarcopenia, and even various types of cancer [2–6]. With regular ex-
ercise training, the defunctionalization due to aging can be partially
recovered leading to medical-cost reduction [2,7]. The mutual promo-
tion between muscle and exercise has been well investigated [8].
Healthy muscle helps maintain a long-term training plan, and exercise
improves the function of muscle. Unfortunately, as age increases, the
mass, strength, and/or performance of muscle decreases. Sarcopenia
might occur during aging [9,10]. Low muscle mass was defined as “pre-
sarcopenia”, which is a superior indicator of functional decline with
aging [11]. Whereas other analysis regards muscle strength a
meaningful metric for muscle health [12–14]. European Working Group
on Sarcopenia in Older People (EWGSOP) has recently updated the
operational definition of sarcopenia, where the emphasis is shifted to-
wards muscle strength [15,16]. The primary importance for body ac-
tivity based successful aging is guaranteeing muscle strength.
Most studies focus on the impact of short-term (weeks or months)
exercise training on muscle strength [17–19]. Typically, just a few
weeks of exercise training can significantly improve muscle strength
and muscle mass. The morphosis of muscle changes accordingly, such
as hyperplasia of myofibers [20]. However, for elderly individuals who
may probably be suffering from sarcopenia, short-term exercise barely
benefits [21,22]. As such, lifelong exercise is expected to reduce the
occurrence of sarcopenia. In some cohort studies, cardiovascular and
skeletal muscle health can be improved by lifelong aerobic exercise
[23–25]. Some spontaneous wheel running intervention mouse model
studies obtain similar analytical results [14,26]. However, due to the

⁎
Corresponding authors at: School of Physical Education & Health, Minhang Campus, East China Normal University, 500th Dongchuan Rd., Shanghai 200241,
China.
E-mail addresses: marvinzy@163.com (Y. Zhao), jlu@tyxx.ecnu.edu.cn (J. Lu).
1
Yu Zhao and Fei Shen contributed equally.

https://doi.org/10.1016/j.lfs.2020.117603
Received 21 January 2020; Received in revised form 15 March 2020; Accepted 24 March 2020
Available online 30 March 2020
0024-3205/ © 2020 Elsevier Inc. All rights reserved.
 Author's Personal Copy
Y. Zhao, et al.
difficulties in tracking, observing, and/or operating, the effect of life-
long treadmill training with rodent model has not been investigated to
the best of authors' knowledge.
Hand grip strength is a suitable indicator for human health and
muscle function [13,27]. In experimental studies, grip strength is also
used to evaluate the variation of muscle function [28–32]. The grip
strength test for rodent model takes the average value of the first three
or five grips as reference [33–36]. However, we observe that, unlike
human test, the difference between grip strength varies dramatically
leading to large variance. The possible reason behind may be that the
rodent cannot follow orders to just grip appropriately with pull
strength.
Hence, we propose a novel rodent model based grip strength test to
investigate the effect of lifelong moderate intensity aerobic treadmill
training on muscle strength, endurance and morphology while aging.
The present study sought to clarify the reliability and validity of the
modified grip strength test. Specifically, in order to detect the effect of
lifelong exercise training on physiological decline in motor function
during aging, the validity of the modified test was investigated.
2. Materials and methods
2.1. Experimental design
First of all, fifty randomly selected adult male BALB/c mice
(24–56 week age) were used to set up a modified grip strength test.
Then, sixty-four male BALB/c mice (6 week age) were used to measure
the influence of lifelong exercise training on muscle. The mice were
assigned into groups Sedentary (n = 32), Exercise (n = 32). We used
the modified grip strength test to measure muscle strength and mor-
phology parameters at the ages of 6, 12, 30 and 64 weeks, respectively.
One side of the gastrocnemius muscle of each mouse was used for
transverse cutting after fixed, and then HE staining, scanning cross-
sectional microscopic image, measuring gastrocnemius cross-sectional
area, myofiber cross-sectional area, myofiber number. The other side of
gastrocnemius muscle was used for isolation and culture of single
myofiber, and the number of myonucleus was counted.
2.2. Experimental animals
BALB/c mice (male, 5 weeks) which are short-lived, were purchased
from Shanghai Lab. Animal Research Center [37]. The mice were
housed in an air-conditioned room with a 12:12 light/dark cycle and
allowed ad libitum access to food and water. All the experiments were
approved by the Animal Care and Use Committee of East China Normal
University and conducted in accordance with the U.S. National In-
stitutes of Health Guide for the Care and Use of Laboratory Animals.
2.3. Treadmill training
Exercise mice were subjected to treadmill training from the age of
6 weeks to 64 weeks at a frequency of 3 times/week. The first week of
the exercise regimen consisted of a training and acclimation period
whereby mice were subject to running beginning at 10.0 m/min for
10 min and gradually increased to 13.3 m/min (0.8 km/h) for 30 min.
In the remainder of the protocol, mice exercised for 30 min at 13.3 m/
min (0% grade). This intensity corresponds to the moderate aerobic
exercise, which is suitable for the lifelong training of mice [38]. All
exercise sessions included brief warm-up and cool-down periods so that
the total treadmill time was approximately 45 min. The sedentary mice
were housed in the same room and were subjected to all identical
conditions, without the exercise training component.
2.4. Grip strength test
A grip strength meter (Bioseb, Model: BIO-GS3, France) was used to
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Life Sciences 251 (2020) 117603
measure the fore and hind limbs grip strength of BALB/c mice. The
strength meter was reset to 0 g after stabilization in each test. Use the
metronome to give a beat of 3 s (3 s is sufficient to prepare for the next
test round and compress the recovery time of the muscle). Consecutive
measurements were performed at the rhythm, which was modified from
previous reports [39]. The mouse's tail was pulled back at a constant
speed after it grasped the grid during a measurement within 3 s. The
peak force in grams was recorded at the time the mouse released its
paws from the grid in each measurement. The test was terminated when
the value of the peak force was lower than 90 g. The order of mice
tested on each day was randomized and the test was only conducted
once within one day. All the tests were operated by one person to en-
sure the reliability.
2.5. Single myofiber culture and fluorescence imaging
Single myofibers were isolated from musculus gastrocnemius of
adult BALB/c mice. Briefly, gastrocnemius muscle were dissected
carefully and subjected to digestion with collagenase I (2 mg/ml,
Sigma, USA) in Dulbecco's Modified Eagle's Medium (DMEM, Sigma,
USA) for 1.5 h at 37 °C. Digestion was stopped by carefully transferring
gastrocnemius to a pre-warmed Petri dish (60 mm) with 6 ml of DMEM
and single myofibers were released by gently flushing muscles with
large bore glass pipette. Released single myofibers were then trans-
ferred and cultured in Matrigel-coated (BD Biosciences, USA) 24-well
culture plates in DMEM supplemented with 3% fetal bovine serum
(FBS, Gibco, USA), 7% horse serum (HS, Gibco, USA), and 1% peni-
cillin-streptomycin (Gibco, USA) for 24 h at 37 °C [40].
After fixing with 2% paraformaldehyde for 10 min, the myofibers
were incubated with 4′,6-diamidino-2-phenylindole (DAPI) for 10 min,
and taken to microscopic imaging by fluorescent microscopy.
2.6. Histopathology
Musculus gastrocnemius was isolated and fixed in 10% neutral
buffered formalin for approximately 24 h, processed and embedded in
paraffin. Embedded tissues were cut into 4–6 micron sections and
stained with hematoxylin and eosin. Stained tissue sections were ex-
amined microscopically and scanned whole transverse section of gas-
trocnemius muscle for statistics.
2.7. Statistical analysis
Data were compared with one-way analysis of variance (ANOVA),
with all ANOVA tests followed by an unpaired t-test, as appropriate.
Normal distribution of data was analyzed by Kolmogorov-Smirnov
normality test. Bonferroni's correction for multiple comparisons was
used. Differences were considered statistically significant when
P < 0.05. Data were assessed using Prism 6.0 (Graphpad Software,
CA). Data were expressed as mean ± standard error of mean (SEM)
and statistical analysis was conducted using SPSS 23.0 (SPSS, Inc.,
USA).
3. Results
3.1. A modified grip strength test and analytical method
We randomly sampled and observed the dynamics of grip strength
during detection in adult BALB/c mice (24–56 week age, n = 50). The
grip strength was measured at 3 second interval. The levels of grip
strength decreased over time in an exponential pattern
(Y = 41.75 × exp(−0.0274 × X) + 98.45) (Fig. 1A–B). Since the
plateau of the curve was near 100 g, a new parameter (Grip
No.(strength > 100g)) was introduced to evaluate the endurance of the
mice, which was defined as the number of the grips before the strength
drops below 100 g during a test.
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Y. Zhao, et al.
Fig. 1. The temporal dynamics of grip strength in adult mice.
A. Typical temporal changes of fore and hind limbs grip strength during a test. B. The levels of grip strength decreased over time in an exponential pattern. The
strength levels from 4 to 15 grips (9–42 s) were shown in right (range of modified grip strength test). Data are mean ± SEM. C–F. Histograms and Gaussian fittings of
Grip No.(strength > 100g) (C), Y0 (D), slope (E) and average value of grip strength (F) calculated from the strength levels from 4 to 15 grips. n = 50.
Gaussian fitting to Grip No.(strength > 100g) histogram revealed that
the levels of Grip No.(strength > 100g) in adult mice was centred at 29.2
with a full width at half maximum (FWHM) of 24.7 (Fig. 1C). Specifi-
cally, the first 3 grips (0–6 s) during a test were highly variable. This
range of value was within the scope of traditional grip strength test
(average of the first 3–5 measures, red rectangle of Fig. 1B). And,
modified test of the strength levels from 4 to 15 grips (9–42 s) displayed
a significant linearity (the right subgraph of Fig. 1B). To simplify the
calculation, the data from 4 to 15 grips were used for further analysis.
Following a linear fitting of the data from 4 to 15 grips, the Y0, slope,
and the average strength were calculated. The Y0 indicating the fitted
maximum value of grip strength was centred at 134.3 g with an FWHM
of 50.2 g (Fig. 1D). The slope representing the change of grip strength
over time was centred at −1.145 g/s with an FWHM of 3.58 g/s
(Fig. 1E). The average grip strength representing the average value of
grip strength was centred at 119.2 g with an FWHM of 32.0 g (Fig. 1F).
The results show that the modified method provides some important
parameters for grip strength as well as some interesting observations.
The modified method is expected to be an efficient substitute in in-
vestigating the possible effect of lifelong exercise training on muscle
function during aging.
3.2. Effect of exercise training on muscle strength while aging
To investigate the effects of lifelong exercise training on muscle
mass and strength in mice, a lifelong treadmill training protocol was
designed. Muscle strength, mass and some morphological parameters
were measured at the ages of 6 (the beginning of the training), 12, 30
and 64 weeks (Fig. 2A).
For muscle strength, the characteristics of the grip strength at dif-
ferent ages were different (Fig. 2D–G), the grip strength was largely
age-dependent. In addition, the lifelong exercise training showed dif-
ferent effects at different ages, and its effects were more significant as
age increased. The best muscle strength was observed at the age of
12 weeks, and its performance decreased gradually during aging
(Fig. 2B–J). Specifically, lifelong exercise training increased the grip
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Life Sciences 251 (2020) 117603
strength and grip strength/body weight ratio at age of 30 and 64 weeks
in mice (Fig. 2C and I), and it increased the Grip No.(strength > 100g) at all
ages detected (Fig. 2B). For the slope, it was worth noted that most of
the values of slope were negative in mice, but the values of the slope
could be positive and were near 0 at the age of 12 weeks, suggesting
that the capacity maintaining a constant grip strength was highest at
the age of 12 weeks (Fig. 2E and H). The lifelong exercise showed no
significant effect on the values of slope in mice (Fig. 2H), but it in-
creased the values of Y0 at the ages of 30 and 64 weeks in mice (Fig. 2J).
These results suggested that lifelong exercise training enhanced muscle
strength in aging.
3.3. Effect of exercise training on muscle morphology while aging
The lifelong treadmill training showed no significant effect on body
weight of the mice (Fig. 3A) but delayed the muscle loss during aging
(Fig. 3B–G). Although lifelong exercise training showed no effect on
gastrocnemius muscle mass at age of 12 and 30 weeks, it increased the
muscle mass at the age of 64 weeks (Fig. 3B). For musculus gastro-
cnemius, the number of myofibers remained constant during aging, but
the cross-sectional area of myofibers decreased during aging
(Fig. 3D–G). Lifelong exercise did not increase the number of myofibers
but increased its cross-sectional area during aging (Fig. 3E–G). These
results suggested that lifelong exercise delayed muscle wasting during
aging by enlarging the cross-sectional area of the myofiber.
3.4. Analysis based on myonuclei
Freshly isolated gastrocnemius myofibers were used for quantifica-
tion of their myonuclei content by DAPI counterstaining (Fig. 4A). The
myonuclear number of sedentary group decreased in two stages, 6 to
12 weeks and 30 to 64 weeks, respectively. The number was stable in
adulthood (12 to 30 weeks). The myonuclear number for exercise group
peaked at 12 weeks and exceeded that for sedentary group (Fig. 4B).
The length of gastrocnemius myofiber barely varied. Myofiber cross-
sectional area/myonuclear number can be used to estimate the changes
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Y. Zhao, et al. Life Sciences 251 (2020) 117603

Fig. 2. Lifelong exercise training improves muscle function.

A. The experimental design of lifelong treadmill training. B. Grip No.(strength > 100g) (muscle force endurance) C. Average value of grip strength. D–G. The dynamics of
fore and hind limbs grip strength measured at ages of 6 (D), 12 (E), 30 (F) and 64 (G) weeks using the modified grip strength test in sedentary and exercise mice. H–J.
(H) Slope, (I) grip strength/body weight and (J) Y0 in sedentary and exercise mice during aging. Data are mean ± SEM. Sedentary group: 6 weeks (n = 8), 12 weeks
(n = 8), 30 weeks (n = 8), 64 weeks (n = 8); Exercise group: 6 weeks (n = 8), 12 weeks (n = 8), 30 weeks (n = 8), 64 weeks (n = 8). *P < 0.05; **P < 0.01.

in relative value of myonuclear domain [41]. Though the myonuclear
domain for both exercise and sedentary group maximized at 12 weeks,
we observed that the myonuclear domain of exercise group was always
larger while aging (Fig. 4C). Lifelong exercise training helped maintain
the myonuclear number and myonuclear domain at a higher level
during aging.
4. Discussion
We apply periodical testing with short periods to effectively im-
prove accuracy and curve fitting to investigate the biological meaning
behind mathematical models. Although the details varies with subjects,
the fundamental steps can be summarized as:
1. Measure the grip strength with short period until the result is less
than predetermined threshold, and count the Grip No.
2. Analyze the dynamics of the grip strength and fit the decay curve.
3. Obtain the optimal parameters (i.e. average value, Y0, slope) with
the most stable portion of grip.
We find the first 3 grips (0–6 s, Fig. 1B) during a test are highly
variable iterations to learn and accommodate the movement with all
four paws while testing. At the beginning of test, the variation is larger
since the mouse is over-forced due to fear or mis-operation. After the
stable range including several grips, the variation increases again be-
cause of the fatigue and impatience (Fig. 1B). Some studies improved
methods by increasing the test intervals, repetition times, or changing
the direction of force measurement [39,42,43]. Our modified test
eliminate the first three grips, instead taking 12 consecutive measure-
ments to reduce the average error. Finally, the trend of changes is
continuous measured and statistically analyzed, which provide us more
insights. Although the traditional test method is more convenient, our
modified grip strength test with more parameters is more preferable as
an animal model for the exercise research.
The modified method provides more measurement parameters.
Besides the average value, Grip No.(strength > 100g), defined as the
number of the grips before the strength level dropping below 100 g
during a test, reflects the strength, as well as the endurance of the
muscle force. Slope reflects the combination of the endurance and the
adaptive capacity of muscle performance during a test. Y0 reflects a
fitted maximum grip strength, which is much similar to the average
value measured by traditional methods. The modified grip strength test

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Y. Zhao, et al. Life Sciences 251 (2020) 117603

Fig. 3. Lifelong exercise delayed the muscle loss in aging.

A. Lifelong exercise showed no significant effect on body weight of the mice.
B–C. The effect of lifelong exercise training on (B) gastrocnemius muscle mass, (C) gastrocnemius muscle mass/body weight. D. Typical gastrocnemius muscle
morphology as detected by H&E staining, (E) the number of gastrocnemius myofibers, (F) myofiber cross-sectional area and (G) the density of myofibers. Data are
mean ± SEM. Sedentary group: 6 weeks (n = 8), 12 weeks (n = 8), 30 weeks (n = 8), 64 weeks (n = 8); Exercise group: 6 weeks (n = 8), 12 weeks (n = 8),
30 weeks (n = 8), 64 weeks (n = 8). *P < 0.05; **P < 0.01.

provides more useful parameters and improves the accuracy of the re-
sults.
Lifelong exercise is a relatively new and evolving area of study with
information. Some studies have suggested that lifelong aerobic exercise
can improve cardiovascular health in the elderly, but the effect of
lifelong exercise on skeletal muscle is still lack of comprehension
[44–47]. Therefore, we carried out the observation and study on life-
long exercise training, and focus on the effect of muscle strength on
preventing muscle atrophy on aged mice. To our knowledge, aerobic
exercise can help control or lose weight, while leading to a loss of lean
body mass [48]. Surprisingly, our study found that lifelong moderate
aerobic training can help maintain weight and muscle mass, as well as
muscle strength in elderly mice. This may be due to the fact that ex-
ercise promotes the development of skeletal muscle in growing mice
and the maintenance of muscle structure and function by long-term
exercise.
While observing and comparing the parameters (Grip
No.(strength > 100g), grip strength, myofiber cross-sectional area) of mice
at 6 weeks, 12 weeks, 30 weeks, and 64 weeks, with and without life-
long treadmill training, the peak value appears at 12 weeks, in this

Fig. 4. Effect of exercise training on myonuclear number and myonuclear domain while aging.
A. Gastrocnemius myofiber from a young exercised male mouse, myonuclei counterstained with DAPI (blue spots). B. The average number of myonuclei in a single
myofiber changes during aging. Data are mean ± SEM. C. The myonuclear domain changes in sedentary and exercise mice during aging. Data are mean value.
Sedentary group: 6 weeks (n = 8), 12 weeks (n = 8), 30 weeks (n = 8), 64 weeks (n = 8); Exercise group: 6 weeks (n = 8), 12 weeks (n = 8), 30 weeks (n = 8),
64 weeks (n = 8). *P < 0.05; **P < 0.01. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

5
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Y. Zhao, et al.
phase, the strength grows mainly because of body development. We
also observe from Fig. 2B that the difference in force endurance be-
tween exercise group and sedentary group is larger since maintaining
force endurance depends more on regular exercise. After 12 weeks, the
grip strength of sedentary group monotonically decreases, while that of
exercise group remains a relatively high level even slightly increases at
aged period (Fig. 2B–C, I). The observation shows that lifelong regular
exercise training is helpful in keeping muscle function while aging.
As for the muscle morphology, declines in muscle mass and myo-
fiber cross-sectional area are observed in aging mice, where the number
of myofibers remains invariant. The exercise shows no significant effect
on the number of myofibers, but decelerates the loss of muscle mass and
the myofiber cross-sectional area during aging, implying that exercise
improves muscle mass through myofiber hyperplasia. Interestingly, the
variation of muscle mass and that of myofiber cross-sectional area do
not strictly follow the variation of muscle strength (Figs. 2C, 3B and F).
The observation indeed supports the muscle strength based operational
definition of sarcopenia [15].
The mature skeletal muscle cells (myofibers) are syncytial and can
contain hundreds of nuclei. “myonuclear domain hypothesis” shows
that a given nucleus controls a defined volume of cytoplasm, so when
muscle grows, the number of myonuclei increases which is donated by
satellite cell [49–51]. When muscle shrinks, whether the number of
myonuclei changes is still unclear. But in vivo time-lapse imaging of
labeled mouse muscle fibers model, the myonuclei barely changes
during muscle atrophy, steady number of myonuclei may be related to
the muscle memory [52,53]. Based on our research on lifelong exercise
training, myofiber tapers and the number of myonuclei decreases while
aging, and myonuclear domain decreases at a faster rate. The ob-
servation contradicts to myonuclear domain hypothesis. The overall
effect of exercise training on myonuclear number and myonuclear do-
main benefits muscle growth in adulthood due to muscle memory.
Although lifelong treadmill training alleviates muscle atrophy, it
cannot avoid the loss of muscle mass during aging (Fig. 3B, C, F). The
size of myosin heavy chain (MHC) Type 2 fiber (fast-twitch) decreases
more with aging, this may result from a decline in capillary density and
blood flow with age [24,54,55]. However, long-term aerobic exercise
training still maintains muscle strength and endurance (Fig. 2B–C),
because aerobic exercise training results in enhanced MHC Type 1 fiber
(slow-twitch) power primarily by increasing force production [24].
Decreases in muscle quantity and quality may occur before the
fourth decade and gradually worsen throughout the later stages of
human [55]. We found that lifelong aerobic exercise training shows
beneficial effects on both muscle mass and muscle strength in aging,
which is consistent with previous studies [14,56]. Exercise training
while body development greatly improves muscle endurance and in-
creases cross-sectional area of myofibers, and the number of myonuclei.
Additionally, regular exercise training in adulthood helps maintain
muscle strength at a relatively high level. Lifelong training is a pre-
ferable exercise prescription in sarcopenia prevention.
However, whether lifelong exercise exerts similar beneficial effects
as short-term exercise needs further investigation. In addition, the
beneficial effects of exercise largely depend on the modalities (in-
tensity, duration, frequency). Thus, more studies are needed to explore
the lifelong exercise's beneficial effects.
5. Conclusion
In this work, we proposed a novel method to investigate the life-
long treadmill training on muscle function while aging. The analysis
was carried out from a multi-parameter perspective. We found that
lifelong exercise training is promising solution to decelerate muscle loss
and helpful in preventing sarcopenia while aging. The proposed method
is an efficient substitute in investigating the complicated relationship
among aging, exercise training, and muscle function.
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Life Sciences 251 (2020) 117603
Declaration of competing interest
The authors declare no competing interests.
Acknowledgements
We thank the Key Laboratory of Adolescent Health Assessment and
Exercise Intervention Ministry of Education, East China Normal
University for supporting this study.
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