Journals of Gerontology: Biological Sciences

cite as: J Gerontol A Biol Sci Med Sci, 2020, Vol. 75, No. 6, 1079–1088
doi:10.1093/gerona/glz142
Advance Access publication June 1, 2019

Original Article

Superior Aerobic Capacity and Indices of Skeletal Muscle

Morphology in Chronically Trained Master Endurance

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Athletes Compared With Untrained Older Adults
James McKendry, PhD,1 Sophie Joanisse, PhD,1,2 Shanat Baig, MBBS,3 Boyang Liu,
MBBS,3 Gianni Parise, PhD,2, Carolyn A. Greig, PhD,1,4,5 and Leigh Breen, PhD1,4,5,*
School of Sport, Exercise & Rehabilitation Sciences, University of Birmingham. 2Department of Kinesiology, McMaster University,
1

Hamilton, Ontario, Canada. 3University Hospital Birmingham NHS Foundation Trust. 4NIHR Birmingham Biomedical Research Centre,
University Hospitals Birmingham NHS Foundation Trust and University of Birmingham. 5MRC-Arthritis Research UK Centre for
Musculoskeletal Ageing Research, University of Birmingham.

*Address correspondence to: Leigh Breen, PhD, School of Sport, Exercise and Rehabilitation Sciences, MRC-Arthritis Research UK Centre for
Musculoskeletal Ageing Research, University of Birmingham, Edgbaston B15 2TT, UK. E-mail: [email protected]

Received: March 29, 2019; Editorial Decision Date: May 26, 2019

Decision Editor: David Le Couteur, MBBS, FRACP, PhD

Abstract
The study aim was to comprehensively assess physiological function and muscle morphology in chronically trained older individuals against
untrained young and older individuals. In a cross-sectional design, 15 young untrained controls (YC) (20 ± 2.7 years, 78.9 ± 13.3 kg), 12
untrained older controls (OC) (69.8 ± 4.1 years, 77.5 ± 14.2 kg), and 14 endurance-trained master athletes (MA) (67.1 ± 4.1 years, 68.7 ±
6.5 kg) underwent assessments of body composition, aerobic capacity, strength, muscle architecture, and fiber-type morphology. Skeletal muscle
index was lower and body fat greater in OC versus MA. Estimated VO2max (mL·kg−1·minute−1) was similar between MA and YC, but lower in
OC. Isometric leg strength normalized to fat-free mass was similar between groups, whereas normalized isometric arm strength was greater
in YC than MA. Myosin heavy chain (MHC) I fiber area was greater in MA than OC, while MHC II fiber area was greater in YC than OC.
MHC II fiber myonuclear domain size was greater in YC than OC and MA, whereas MA had greater MHC I and MHC II fiber capillarization
than OC and YC. Satellite cell content was similar between groups. Chronic endurance training enhances indices of muscle morphology and
improves body composition and aerobic capacity in older age, with potentially important implications for healthspan extension.

Keywords: Sarcopenia, Exercise, Muscle, Human ageing

Introduction energy storage, and nutrient deposition (5). By the eighth decade of
life, skeletal muscle mass has reduced by ~18% in men and 27% in
The shift toward an ageing population presents a significant and
women (6) and is accompanied by a greater relative loss of muscle
overwhelming global demand on health care resources. The major
strength (7). Sarcopenia is characterized by reductions in muscle fiber
trepidation is not that individuals are living longer (ie, life span), but
cross-sectional area (CSA) (8), satellite cell content (9), motor unit
that they endure a larger portion of their later years with multiple age-
remodeling (10), infiltrations of fat and connective tissue (11), alter-
associated comorbidities (ie, healthspan) (1). Thus, strategies to close
ations to the microcirculation (9,12), and reduced oxidative capacity
the gap between health- and life-span are of paramount importance.
(13). The extent to which this physiological deterioration is due to
Age-related reductions of skeletal muscle mass, strength and func-
inherent ageing processes, free from artifacts of biological ageing that
tion (termed “sarcopenia”), and cardiorespiratory fitness may pre-
exacerbate the decline (ie, physical inactivity), is unclear.
maturely force individuals into a state of physical dependence and
The study of individuals who have chronically undertaken
are independent predictors of all-cause mortality (2–4). Skeletal
structured exercise training and continue to compete, into their
muscle mass is indispensable for locomotion, basal metabolism,
later years, referred to as master athletes (MA), provides a model to
© The Author(s) 2019. Published by Oxford University Press on behalf of The Gerontological Society of America.
1079
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1080 Journals of Gerontology: BIOLOGICAL SCIENCES, 2020, Vol. 75, No. 6

investigate the impact of inherent ageing on physiological function, Methods

without confounding aspects of biological ageing. In a recent meta-
Participants
analysis, we reported that endurance-based MA displayed a 55%
greater aerobic capacity compared with age-matched untrained Fifteen untrained young men (YC) and 12 older untrained men
individuals, whereas resistance trained MA demonstrated greater (OC) were recruited alongside 14 master endurance-trained men
strength than age-matched untrained individuals (14). Furthermore, (MA) through local advertisements, the British Masters Athletics
others have shown that although a decline in physiological func- Federation, and the League of Veteran Racing Cyclists. Young (18–
tion remains apparent in MA, high physical activity may ultimately 35 years) and older untrained controls (60–80 years) were deemed
improve the healthspan by shifting the “set point” of age-related eligible for study participation only if they maintained habitual ac-
physiological deterioration to later in life (13,15). Nevertheless, tivity and had not previously participated in any form of structured
firm conclusions on the influence of ageing and chronic exercise on exercise training outside of recreational activities. MA (60–80 years)
were included only if they had maintained continuous endurance

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physiological deterioration cannot be drawn, as very few studies
have included sufficient parameters to comprehensively charac- training at least twice per week for ≥20 years preceding the study.
terize skeletal muscle morphology at macro- and microscopic level Exclusion criteria included regular consumption of analgesic or
in MA (16–18), or have not included a young control group for anti-inflammatory drug(s), prescription or non-prescription, that
comparison (12,13). The few studies that have investigated aspects may affect muscle metabolism (eg, beta-blockers, corticosteroids,
of MA physiology and muscle morphology report conflicting find- non-steroidal anti-inflammatories). Participant anthropometric and
ings, which may relate to different analytical methods or the specific training characteristics are detailed in Table 1. All participants were
muscle studied. Specifically, some have demonstrated superior leg informed of the purpose and methodology of the study, were deemed
strength and muscle fiber diameter in senior sportsmen compared healthy by completion of a general health questionnaire assessment,
with untrained older individuals (17), whereas others have shown and provided their written informed consent. Ethical approval was
similar muscle size, strength, and fiber CSA between MA and un- obtained through the West Midlands – Solihull Research Ethics
trained older individuals (16,18). Committee (16/WM/0167). The study conformed to the standards
Therefore, to better understand the role of inherent ageing pro- set by the Declaration of Helsinki (seventh version).
cesses in physiological function, and the extent to which chronic
exercise training might improve the healthspan, the aim of the cur- Study Design
rent study was to conduct the most comprehensive comparison, to In a parallel study design, YC, OC, and MA were recruited to inves-
date, of physical function, cardiorespiratory fitness, body compos- tigate the effect that ageing and continuous endurance exercise exert
ition, muscle strength, architecture, and fiber-type morphology be- on indices of muscle mass, function, and morphology. Following
tween MA and healthy untrained younger and age-matched older initial study screening and consenting, participants reported to the
individuals. We hypothesized that untrained older individuals would School of Sport, Exercise and Rehabilitation Sciences (SportExR)
exhibit an impairment in all of the above parameters compared with laboratory on two separate occasions with each visit separated by
younger individuals, which would be partially or, in some cases, ~7 days. For each visit, participants reported to SportExR in an over-
completely absent in MA. night fasted-state, having refrained from strenuous physical activity

Table 1. Participants’ Anthropometric and Training Characteristics

YC (N = 15) MA (N = 14) OC (N = 12)

Age (years) 20.0 ± 2.7**,## 67.1 ± 6.4 69.8 ± 4.1

Height (m) 1.80 ± 0.04* 1.70 ± 0.06 1.80 ± 0.07
Body mass (kg) 78.9 ± 13.3 68.7 ± 6.6 77.5 ± 14.2
BMI (kg·m−2) 24.6 ± 3.6 23.0 ± 2.0 24.5 ± 3.8
Whole-body FFM (kg) 56.9 ± 6.6 52.2 ± 3.5 52.9 ± 7.8
Whole-body FM (kg) 17.6 ± 7.4 13.3 ± 3.9# 20.9 ± 7.1
Body fat (%) 22.0 ± 5.5 19.2 ± 4.1## 26.8 ± 5.4
Skeletal muscle index (%) 74.0 ± 5.2 77.0 ± 4.0## 69.8 ± 5.1
ALM/height2 (kg·m−2) 8.18 ± 0.79# 7.71 ± 0.54 7.39 ± 0.95
Systolic blood pressure (mm Hg) 126 ± 9 125 ± 7 137 ± 18
Diastolic blood pressure (mm Hg) 64 ± 8 76 ± 7 83 ± 11
Fasting plasma glucose (mmol·L−1) 5.33 ± 0.39## 5.35 ± 0.63## 5.96 ± 0.35
Fasting serum insulin (µIU · mL−1) 10.95 ± 3.24* 7.21 ± 3.10 11.42 ± 4.38*
HOMA-IR 2.59 ± 0.75** 1.65 ± 0.61 3.05 ± 1.16**
CRP (mg·L−1) 1.71 ± 1.89 0.77 ± 0.43 1.10 ± 0.53
Training experience (years) — 36.5 ± 8.1 —
Training frequency (sessions·week−1) — 4.5 ± 1.4 —
Training duration (hours·week−1) — 7.6 ± 4.7 —
Training distance (km·week−1) — 210±12 / 50 ±15 —

Note: ALM = appendicular lean mass; BMI = body mass index; CRP = C-reactive protein; FFM = fat-free mass; FM = fat mass; HOMA-IR = Homeostatic Model
Assessment of Insulin Resistance. MA = old endurance-trained master athletes; OC = old untrained individuals; YC = young untrained individuals. Data presented
as mean ± SD. Training distance is separated into cyclists (n = 8, left) and runners (n = 6, right).
*Significantly different from MA, p <.05. **Significantly different from MA, p <.01. #Significantly different from OC, p <.05. ##Significantly different from OC,
p <.01.
Journals of Gerontology: BIOLOGICAL SCIENCES, 2020, Vol. 75, No. 6 1081

and alcohol for at least 48 hours previously, and from caffeine con- Where ΔHR is the difference in the average heart rate between the
sumption on the day of the trial. During the initial visit, participants two workloads, ΔPO is a constant reflecting the difference in power
underwent assessments of body composition, aerobic capacity, max- output between the two workloads, where sex is 0 = woman and
imal limb strength, and a battery of functional tests. Approximately 1 = man, and age is participants age in years as an integer.
1 week later, participants underwent ultrasound scanning, a single
venous blood sample, and a single muscle biopsy. Short Physical Performance Battery
Participants completed the Short Physical Performance Battery
Visit 1 (SPPB) of balance, gait speed, and low limb power (21). The balance
Body mass, height, and composition test comprised three increasingly difficult stances (side-by-side, semi-
Body mass was determined by weighing each participant in loose tandem, full tandem), which participants were required to hold for
clothing, without shoes, to the nearest 0.1 kg using a digital balance 10 seconds. For the gait speed test, participants were instructed to

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scale (Esca 813, Hamburg, Germany). Height measurements were walk a 3-meter-long course “at your usual speed as if walking to the
made to the nearest 0.1 cm using a stadiometer (Seca 217, Hamburg, shops.” Finally, participants completed a repeated chair stand test,
Germany). Participants underwent a dual-energy x-ray absorpti- in which they were instructed to rise from a chair, with arms folded,
ometry (DXA) scan (Discovery DXA Systems, Hologic Inc., Bedford, five times as quickly as possible. The result from each of the three
MA) to determine whole-body and regional fat- and fat-free mass tests were scored out of 4 marks and compared against predeter-
(FFM). Skeletal muscle index was calculated as FFM as a percentage mined criteria for a score of physical function.
of whole-body mass.
Handgrip and leg strength
Blood pressure Individuals stood, feet side-by-side, with their arms adducted,
Blood pressure was measured using a standard fully automatic blood wrists neutrally rotated and the dynamometer (Jamar Hydraulic,
pressure monitor (OMRON M2, OMRON Healthcare UK Ltd., Patterson Medical, UK) positioned comfortably in the participant’s
UK). Participants were asked to remove any clothing that obstructed self-reported dominant hand. Participants were instructed to squeeze
the blood pressure cuff. Participants were seated with their legs un- the handle as hard as possible and three attempts were recorded,
crossed and back supported, encouraged to relax and refrain from with the highest value recorded. Maximal knee extension and
talking during the assessment. This test was repeated three times elbow flexion isometric strength were measured using a KinCom
and the lowest of three readings taken. Following blood pressure Dynamometer (KinCom 125AP, KinCom, USA) on the self-reported
assessment, participants underwent the Ekblom-Bak submaximal dominant limb. Participants were seated, with the tested limb, chest
cycle ergometry test for the estimation of V̇O2max (19), the details of and hips stabilized, and with the knee and elbow angle positioned
which are described in the Supplementary Material. at 70 degrees of flexion, respectively. After familiarization, partici-
pants performed three maximal voluntary contractions (MVCs), the
Aerobic capacity greatest of which was recorded.
Participants underwent the Ekblom-Bak test (19), a submaximal
cycle ergometry test for the estimation of V̇O2max . Briefly, the test Dietary analysis
is based on the change in heart rate between a standardized low Between Visits 1 and 2, participants were provided with a 4-day
workload and a higher workload predetermined by sex and current weighed food diary designed to capture habitual food intake on 2 con-
habitual activity. Participants were instructed to maintain a constant secutive weekdays and 2 weekend days. Participants were instructed
cadence of 60 rpm throughout the test. First, the participant cycled not to change their usual diet and to be as accurate as possible when
for 4 minutes, while investigators ensured constant cadence and re- describing the food (cooking method, brand, amount etc.). Dietary
sistance. Heart rate was recorded every 15 seconds and averaged intake was analyzed using MyFitnessPal software (MyFitnessPal Inc.).
during the final minute of the low workload. The electrocardiogram
(ECG) was monitored over 5 minutes prior to and continuously Physical activity
throughout exercise by a cardiologist. The resistance was then in- Between Visits 1 and 2 (~7 days), participants were provided with
creased to the predetermined level for the next 4 minutes, while ca- a wrist-worn accelerometer (GENEActiv, ActivInsights Ltd., UK)
dence and resistance remained constant. During the second minute designed to capture habitual activity in free-living conditions over
at the higher workload, individuals communicated their rate per- 5 consecutive days (including both weekend days). Accelerometers
ceived exertion (RPE) using a standard scale (20). If RPE was <10, were initialized to sample data at a 10 Hz. Data were converted
the resistance was increased by 1 kp and the second workload tim- into 60-second epochs and analyzed using the GENEActiv software
ings started again. If RPE was 10–11, the workload was increased (version 2.2, ActivInsights). Activities were split into four categories
by 0.5 kp and the second workload timings started again. If RPE based on metabolic equivalent (MET) values: (i) sedentary activity
was 12–16, participants were instructed to maintain this workload. (<1.5 METs), (ii) light activity (1.5–3.99 METs), (iii) moderate ac-
If RPE was >17, the exercise bout was stopped. Heart rate was re- tivity (4.0–6.99 METs), and (iv) vigorous activity (>7 METs) (22).
corded every 15 seconds and averaged during the final minute of the
higher workload. V̇O2max was calculated using the following equa- Visit 2
tion (19):
Muscle architecture
Å ã B-mode ultrasonography with a linear array probe was used for all
∆HR
V̇O2max = 4.98196 − 2.88618 + 0.65015 (Sex) measurements of muscle architecture. Participants lay fully relaxed in
∆PO (1)
− 0.01712(Age) a supine position with a small towel rolled and placed under the knee
 for anterior measurements. For posterior measurements, participants
1082 Journals of Gerontology: BIOLOGICAL SCIENCES, 2020, Vol. 75, No. 6

were in a fully relaxed prone position with feet overhanging the end 2 hours. Following washes, sections were then re-blocked in 10% goat
of the bed. Biceps brachii architecture was measured with the par- serum in PBS and incubated in a primary antibody cocktail consisting
ticipants seated on the edge of the bed with the arm freely hanging. of myosin heavy chain (MHC) I (A.4.951; slow isoform) and laminin.
The muscles measured included; vastus lateralis, vastus intermedius, Sections were washed and then incubated in a secondary antibody
rectus femoris, biceps brachii, gastrocnemius medialis, and tibialis cocktail, and nuclei were labeled with DAPI (49,6-diamidino-2-
anterior. Measurements of muscle thickness (MT), pennation angle phenylindole) (1:20000, Sigma-Aldrich, Oakville, Ontario, Canada)
(θ), and fascicle length (Lf) were made in triplicate. MT was con- prior to cover slipping with DAKO fluorescent mounting media
sidered to be the distance between deep and superficial aponeuroses. (Burlington, Ontario, Canada). Slides were visualized with the Nikon
θ was calculated as the angle between the muscle fascicle and the Eclipse Ti Microscope (Nikon Instruments, Inc., Melville, NY, USA),
deep aponeurosis. Fascicle length was measured as the length of the equipped with a high-resolution Photometrics CoolSNAP HQ2
fascicular path between the two aponeuroses. Measurement sites fluorescent camera (Nikon Instruments). Images were captures and

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were set at 60% of the upper arm length distal to the acromion analyzed using the Nikon NIS Elements AR 3.2 software (Nikon
process for biceps brachii, 50% of the muscle length for upper thigh Instruments). All images were obtained on the ×20 objective.
muscles, and 30% of the leg length distal to the popliteal crease
for lower leg muscles. The ultrasound probe was covered with Muscle fiber type and CSA
water-soluble transmission gel, to provide acoustic contact without This analysis was carried out on a subset of participants (YC:
compressing the dermal surface, and an image was taken when a n = 13; MA: n = 14; OC: n = 11). Serial 10-µm sections of skeletal
number of fascicles could clearly be identified. muscle biopsies were cut in the cryostat at −20°C. Briefly, slides were
washed in Triton x-100 (0.02%) to permeabilize the fibers. Slides
Blood sample were blocked for 90 minutes in 5% normal goat serum (Invitrogen,
A blood sample was obtained via venepuncture from an UK) and then incubated overnight in the primary antibody cocktail:
antecubital forearm vein. Blood was collected in separate MHC I, BAF8 (Developmental Studies Hybridoma Bank [DSHB],
ethylenediaminetetraacetic acid (EDTA) and serum-separating USA); MHC II, SC-71 (DSHB); laminin in PBS. Slides were washed
polymer gel containing BD vacutainers (BD, Oxford, UK). Blood sam- in 3 × 5 minutes in PBST and incubated in the secondary antibody
ples were centrifuged at 3,000g for 10 minutes at 4°C, and serum and cocktail. Slides were then washed and mounted with coverslips in
plasma aliquots were frozen at −80°C for later analysis. prolong gold anti-fade mountant (Thermo Fisher Scientific, Paisley,
UK). Prepared slides were observed under a Nikon E600 microscope
Muscle biopsy using a 20 × 0.75 numerical aperture objective. Images per area were
A muscle biopsy sample was obtained from the quadriceps vastus captured under two color filters achieved by a SPOT RT KE color
lateralis under local anesthesia (1% lidocaine) using the Bergström three-shot CCD camera (Diagnostic Instruments Inc., MI, USA), il-
needle technique (23). Muscle biopsy tissue was quickly rinsed in luminated by a 170 W Xenon light source. Texas red (540–580 nm)
ice-cold saline and blotted to remove any visible fat and connective excitation filter was used to capture MHC I images and FITC (465–
tissue before being frozen in liquid nitrogen or placed in a pipette tip 495 nm) excitation filter was used to capture MHC II and laminin.
with optimum cutting temperature compound (Tissue-Tek® [O.C.T.] Microscope slides were prepared such that each slide contained two
Compound, Sakura® Finetek) and frozen in liquid nitrogen-cooled serial sections from one individual from each of the included groups.
isopentane and stored at −80°C for later analysis. Images were captured, and measured, such that ~600–1,000 muscle
fibers were included for analysis from each group. All viable muscle
Sample Analysis fibers in any particular image, excluding those displaying freeze frac-
ture artifact and any longitudinal fibers (assessed as those with a
Blood samples
circularity of <0.6), were included for analysis and were analyzed
Plasma glucose was analyzed using a commercially available blood
using Image J Fiji (25).
glucose analyzer (HemoCue® Hb 201+ System, HemoCue AB,
Sweden). Serum insulin and C-reactive protein (CRP) were ana-
lyzed using commercially available enzyme-linked immunosorbent Calculations and measurements
assay (ELISA) kits (IBL International GmbH, Hamburg, Germany) Myonuclear domain
following the manufacturer instructions. Postabsorptive insulin sen- This analysis was carried out on a subset of participants (YC: n = 10;
sitivity was also estimated using the homeostasis model of insulin MA: n = 12; OC: n = 11). From acquired images, specific channels
resistance index (HOMA-IR) (24). were extracted so that only MHC I, laminin, and DAPI were visual-
ized. Myonuclear domain was determined as the fiber area of MCH
Immunohistochemistry I and MHC II fibers (µm2) per nucleus. Only nuclei that were lo-
Detailed information on primary and secondary antibodies for cated within the basal lamina as identified by laminin were counted.
immunohistochemistry is presented in Supplemental Table 1. For ana- Myonuclear content was determined by counting the number of nu-
lysis of myonuclear domain, Satellite cell (SC) content and myofiber clei residing within the basal lamina of either Type I (MHC I posi-
capillarization muscle cross-sections were stained as follows. Briefly, tive) or Type II (MHC I negative) fibers expressed in relation to total
tissue sections were fixed in 4% paraformaldehyde (PFA) for 10 min- MHC I and MHC II fibers.
utes, washed 3 × 5 minutes in phosphate-buffered saline with Tween
20 (PBST), blocked for 60 minutes at RT (in PBS containing 2% bo- Myofiber capillary analysis
vine serum albumin, 5% fetal bovine serum (FBS), 0.2% Triton x-100, This analysis was carried out on a subset of participants (YC: n = 10;
0.1% NaAzide, and 2% goat serum) then incubated overnight in pri- MA: n = 11; OC: n = 11). From acquired images, specific channels
mary antibodies specific for Pax7 and CD31 at 4°C. Following washes, were extracted so that only MHC I, laminin, and CD31 were visu-
sections were then incubated in appropriate secondary antibodies for alized. Quantification of (i) capillary contacts (CCs, the number of
Journals of Gerontology: BIOLOGICAL SCIENCES, 2020, Vol. 75, No. 6 1083

capillaries around a fiber), (ii) the capillary‐to‐fiber ratio on an indi- Results

vidual fiber basis (C/Fi), (iii) the number of fibers sharing each capil-
Participant Characteristics
lary (ie, the sharing factor), and (iv) the capillary-to-fiber perimeter
Participant anthropometric and training characteristics are detailed in
exchange index (CFPE index, calculated as the C:Fi/P, where P is the
Table 1. YC were taller than MA (p = .031). MA had lower whole-
fiber perimeter) was based on previous published protocols (26,27).
body fat mass (p = .011), a lower body fat percentage (p = .002), and a
greater skeletal muscle index (p = .001) than OC. YC had significantly
Myofiber satellite cell analysis
greater appendicular lean mass than OC (p = .03). YC and MA had
This analysis was carried out on a subset of participants (YC: n = 10;
significantly lower fasting glucose concentrations than OC (p = .005
MA: n = 11; OC: n = 10). From acquired images, specific channels were
and p = .009, respectively). MA had significantly lower serum insulin
extracted so that only MHC I, laminin, Pax7, and DAPI were visual-
(p = .048 and p = .041) and HOMA-IR (p = .03 and p = .003) than
ized. Fiber type-specific SC content was determined by identifying nu-
YC and OC, respectively. There were no other significant differences

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clei co-localized with Pax7 that resided within the basal lamina.
in anthropometric characteristics between the groups.

Statistical Analysis
Physical Function
Data were expressed as mean ± SD. Normality of data was assessed
Physical function characteristics are detailed in Table 2. There were
using the Shapiro–Wilk test. Between-group differences in anthropo-
no significant differences between groups for any of the SPPB com-
metric characteristics, physical performance, dietary intake, and ha-
ponent nor total scores or handgrip strength. YC produced signifi-
bitual activity levels were identified via one-way analysis of variance
cantly greater knee extension MVC than MA and OC (p = .006 and
(ANOVA) analysis with one within-group factor (eg, FFM) and three
p < .001, respectively), although no difference was apparent when
groups for comparison. Muscle morphological data were analyzed
normalized to leg FFM indicating no between-group difference in
via mixed-design ANOVA with one between factor (three levels;
relative force production. YC produced significantly greater elbow
group) and one within factor (two levels; fiber type). Specifically,
flexion than MA (p < .001) that was apparent when normalized to
the average of each participant’s data was incorporated into the
arm FFM (p < .05), indicating lower relative force production in MA
grouped analyses. For example, when analyzing fiber-specific CSA,
versus YC. YC and MA had a significantly greater estimated V̇O2max
~100 fibers were measured for each individual, following which,
than OC (p < .001).
the average fiber CSA for each individual was incorporated into the
grouped analysis. Significant main effects indicated group differences
with fiber types collapsed (ie, MA vs OC vs YC for MHC I and II Muscle Architecture
combined), or fiber-type differences with groups collapsed (ie, MHC Muscle architecture results are detailed in Supplementary Table 2.
I vs MHC II for MA, OC, and YC combined). Significant interaction YC had significantly greater MT than OC for vastus lateralis (p <
effects indicated that age and/or training status (group; YC, MA, OC) .001), vastus intermedius (p = .002), and rectus femoris (p < .001)
influenced a given fiber-type parameter (ie, MHC I CSA). Whenever and greater MT than MA for rectus femoris (p < .001). YC had sig-
a significant F ratio was found (ie, variation between sample means), nificantly greater θ than OC for vastus lateralis (p = .007), vastus
interaction or main effects were followed up with pairwise compari- intermedius (p = .014), and rectus femoris (p = .002) and signifi-
sons using independent t-tests to locate specific differences, with the cantly greater θ than MA for rectus femoris (p = .04). MA had sig-
Bonferroni correction applied to account for multiple comparisons. nificantly greater Lf than OC for gastrocnemius medialis (p = .032)
Significance level was set at p <.05 for all analyses. All statistical ana- and YC had significantly greater Lf than OC (p = .024) and MA
lyses were performed using SPSS version 22.0 (Chicago, IL). (p = .041) for gastrocnemius lateralis.

Table 2. Physical Function and Strength

YC (N = 15) MA (N = 14) OC (N = 12)

SPPB standing balance (second) 10.0 ± 0 10.0 ± 0 10.0 ± 0

SPPB semi-tandem (second) 10.0 ± 0 10.0 ± 0 10.0 ± 0
SPPB full tandem (second) 10.0 ± 0 9.7 ± 1.0 9.7 ± 1.1
SPPB 3-m walk (second) 2.37 ± 0.34 2.43 ± 0.28 2.45 ± 0.44
SPPB 5× sit-to-stand (second) 7.27 ± 1.34 7.97 ± 1.88 7.88 ± 2.03
Handgrip strength (kg) 50.9 ± 7.6 47.0 ± 5.9 46.3 ± 8.4
Knee extension MVC (Nm) 638 ± 82**,## 520 ± 105 471 ± 118
Knee extension MVC (Nm·kg−1 leg FFM) 68 ± 9 62 ± 13 57 ± 13
Elbow flexion MVC (Nm) 293 ± 49** 223 ± 43 257 ± 49
Elbow flexion MVC (Nm·kg−1 arm FFM) 79 ± 9* 68 ± 12 77 ± 10
Estimated V̇O2max (mL·kg−1·minute−1) 53.2 ± 7.3## 49.3 ± 3.6## 36.7 ± 6.5
Estimated V̇O2max (mL·kg−1 FFM−1·min−1) 72.7 ± 5.8##,** 65.2 ± 3.4## 53.3 ± 8.7

Note: FFM = fat-free mass; MVC = maximal voluntary contraction; Nm = Newton meters; SPPB = Short Physical Performance Battery. MA = old endurance-
trained master athletes; OC = old untrained individuals; YC = young untrained individuals. Data presented as mean ± SD.
*Significantly different from MA, p <.05. **Significantly different from MA, p <.01. #Significantly different from OC, p <.05. ##Significantly different from OC,
p <.01.
1084 Journals of Gerontology: BIOLOGICAL SCIENCES, 2020, Vol. 75, No. 6

Habitual Dietary Intake and Physical Activity (p = .04). No significant difference was observed between any of
Dietary intake results are detailed in Supplementary Table 3 (upper). the groups for MHC I fiber myonuclear domain. YC had signifi-
YC total energy (p = .014), fat (p = .019), protein (p < .001), relative cantly greater MHC II fiber-specific myonuclear domain than MA
fat (p = .044), and relative protein intake (p = .005) were greater than (p = .018) and OC (p = .006). No significant interaction effect for
OC. Whereas, MA total energy (p = .003), carbohydrate (p = .014), group*fiber type myonuclei content was observed.
relative carbohydrate (p = .008), relative fat (p < .007), and relative
protein intake (p = .029) were greater than OC. No differences in Capillarization and Satellite Cells
dietary intake were observed between YC and MA. Habitual activity Capillarization and SC data are detailed in Figure 3A–G and
can be found in Supplementary Table 3 (lower). MA carried out sig- Supplementary Figure 1A–C, respectively. A significant interaction
nificantly more vigorous activity (49 ± 29 minutes·day−1 or 5.6 ± effect of group*fiber type CCs was observed (p = .007). MA had sig-
3.2%·day−1) than both YC (13 ± 14 minutes·day−1, 1.7 ± 1.5%·day−1; nificantly more CC per MHC I myofiber compared with YC and OC

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p < .001) and OC (4 ± 6 minutes·day−1, 1.1 ± 2.6% day−1; p < .001). (p = .002 and p < .001, respectively). MA had more CC per MHC II
There were no other differences in physical activity between groups. fiber compared with OC (p = .006). A significant interaction effect
of group*fiber type C/Fi was observed (p = .007). MHC I C/Fi was
Muscle Fiber Properties greater in MA compared with YC and OC (p = .001 and p = .001,
Muscle fiber data are detailed in Figure 1A–E. A significant inter- respectively). MA had a greater MHC II C/Fi than OC (p = .004). No
action effect of group*fiber type CSA was observed (p < .001). MA significant interaction effect of group*fiber type CFPE index was ob-
displayed larger MHC I fiber CSA than OC (p = .031), whereas served. However, CFPE showed a significant main effect for group,
YC had significantly larger MHC II fiber CSA than OC (p = .008). such that MA had a significantly greater CFPE compared with YC
A significant interaction effect of group*fiber type distribution was (p = .001) and OC (p = .001). No significant interaction (group*fiber
observed (p < .001). MA (57 ± 15%) had a significantly greater pro- type) or main effects (group and fiber type alone) were observed for
portion of MHC I muscle fibers compared with YC (35 ± 10%, p < SC content.
.001) and OC (41 ± 10%, p = .008). MA (43 ± 15%) had a signifi-
cantly lower proportion of MHC II fibers compared with YC (65 ±
10%, p < .001) and OC (58 ± 10%, p = .012).
Discussion
Ageing has a deleterious effect on physiological function, muscle
Myonuclear Domain mass, and strength (sarcopenia) and muscle oxidative capacity that
Muscle fiber data are detailed in Figure 2A–E. A significant inter-
action effect of group*fiber type myonuclear domain was observed

Figure 1. Cross-sectional area (CSA) of myosin heavy chain (MHC) I (A) and Figure 2. Myonuclear domain in myosin heavy chain (MHC) I (A) and
MHC II muscle fibers (B) and relative abundance of MHC I (C) and MHC II MHC II muscle fibers (B) and number of nuclei per MHC I (C) and MHC II
muscle fibers (D) in young untrained individuals (YC), old endurance-trained muscle fiber (D) in young untrained individuals (YC), old endurance-trained
master athletes (MA), and old untrained individuals (OC). Representative master athletes (MA), and old untrained individuals (OC). Representative
immunohistochemical image of muscle fiber CSA from a biopsy sample in immunohistochemical image of fiber-type myonuclei from a biopsy sample
YC (left), MA (center), and OC (right) with MHC I fiber MHC in red, MHC II fiber in YC (left), MA (center), and OC (right) with DAPI stained nuclei in blue,
MHC in green, and laminin stained cell membrane in green (E). Significance MHC I fiber MHC in green, and laminin stained cell membrane in green (E).
was set at p <.05. #Significantly different from OC (p < .05) and *Significantly Significance was set at p < .05. ##Significantly different from OC (p < .01).
different from MA (p < .05). Values are presented as the median (central *Significantly different from MA (p < .05). Values are presented as the median
horizontal line), 25th and 75th percentiles (box), minimum and maximum (central horizontal line), 25th and 75th percentiles (box), minimum and
values (vertical lines), and mean (cross). maximum values (vertical lines), and mean (cross).
Journals of Gerontology: BIOLOGICAL SCIENCES, 2020, Vol. 75, No. 6 1085

that highly active MA had a greater skeletal muscle index, lower

body fat, and lower whole-body insulin resistance than OC. Thus,
continuation of structured endurance exercise training promotes a
favorable body composition and could confer important metabolic
health benefits.
Age-related muscle mass and strength loss involve alterations
in architectural properties and fiber-type morphology. With ageing,
muscle fiber fascicle length and pennation angle decrease, indicating
a loss of sarcomeres (in series and parallel) (34). To the best of our
knowledge, we are the first to measure muscle architecture in an
endurance-trained MA cohort, for comparison against YC and OC.

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Our novel findings demonstrate that muscle fiber fascicle length
and pennation angle properties were generally lower in OC and
MA, compared with YC, with no differences between MA and OC.
Similarly, others have reported equivalent vastus lateralis fascicle
length between young and old sprint athletes (35). Important to
note, however, was that fiber pennation angle and fascicle length
of the vastus intermedius and gastrocnemius medialis, respectively,
Figure 3. Capillary contacts per fiber (CC), individual capillary-to-fiber
ratio (C/Fi), and capillary-to-fiber perimeter exchange index (CFPE) in
were statistically greater in YC compared with OC only, with no dif-
myosin heavy chain (MHC) I fibers (A, C, and E, respectively) and MHC II ference between OC and MA. These data indicate that the alterations
fibers (B, D, and F, respectively), in young untrained individuals (YC), old to muscle architectural properties with advancing age are not pre-
endurance-trained master athletes (MA), and old untrained individuals (OC). served with chronic endurance exercise training. The observed archi-
Representative immunohistochemical image of fiber-type capillarization tectural modifications in MA and OC may attenuate the decreased
from a biopsy sample in YC (top), MA (middle), and OC (bottom) with CD31-
force production with age-related muscle loss. Indeed, isometric knee
stained capillaries in purple (denoted by white arrows), MHC I fiber stained in
extensor strength was similar between groups when normalized to
green, and laminin stained cell membrane in green (G). Significance was set
at p <.05. ##Significantly different from OC (p < .01). ††Significantly different leg FFM. Although normalized isometric biceps strength was lower
from YC (p < .01). Values are presented as the median (central horizontal line), in MA compared with YC, technical limitations prevented us from
25th and 75th percentiles (box), minimum and maximum values (vertical understanding whether alterations to the architectural properties of
lines), and mean (cross). this muscle could explain this deficit. Nonetheless, it is apparent that
contractile loading in the form of resistance training may be required
may be exacerbated by low physical activity or adverse change in to generate sufficient mechanical tension to enhance muscle architec-
body composition. Individuals who have chronically undertaken tural properties and mitigate strength declines in older individuals
structured exercise training, or MA, provide an opportunity to (14,36).
study mechanisms of inherent physiological ageing dissociated from Reductions in muscle fiber number and area, particularly MHC
aspects of biological ageing (15). Previously, we have highlighted II fibers, is a characteristic of sarcopenia (8). In older individuals,
the dearth of studies providing a comprehensive characterization resistance training increases MHC II fiber area, whereas endurance
of the MA phenotype against untrained young and older individ- exercise training primarily augments MHC I fiber area (14). Skeletal
uals (14). Herein, we comprehensively address this issue by firstly muscle morphology data in MA are scarce, and available studies
demonstrating that endurance-trained MA display superior body have yielded conflicting findings (14,16,17), making it difficult to
composition (lower body fat and higher skeletal muscle mass index) fully elucidate the mechanisms through which chronic exercise en-
and aerobic fitness compared with untrained age-matched older in- hances physiological function. In line with previous findings (17,37),
dividuals (OC), to a level comparable with untrained healthy young we demonstrate that endurance-trained MA have larger MHC I fi-
individuals (YC). Secondly, we provide novel morphological insights bers than OC, and a greater proportion of MHC I fibers compared
demonstrating that MA display larger MHC I muscle fiber area than with YC and OC. Compared with OC, the superior MHC I fiber
OC. Furthermore, MA display greater capillarization of MHC I and properties of MA suggests that their greater maximal aerobic cap-
II fibers compared with OC and YC. MHC II fiber myonuclear do- acity may be partly underpinned by a greater muscle oxidative po-
main size was greater in YC compared with OC and MA, whereas tential, although cardiorespiratory adaptations undoubtedly also
SC content was similar between groups. Collectively, these data play a key role in VO2max. Unsurprisingly, MHC II fiber size was
suggest that chronic endurance training promotes superior aerobic lower in OC compared with YC. However, chronic endurance exer-
capacity, body composition, and fiber capillarization, with potential cise did not preserve MHC II fiber size, which was similar between
implications for healthspan extension. MA and OC. Due to technical limitations, we were only able to ana-
Sarcopenia is often accompanied, and in some cases masked, lyze two fiber types. Given evidence that ageing leads to a reduction
by increased adiposity (28) and ectopic and visceral fat deposition in MHC IIA fibers, an increased proportion of fibers co-expressing
(11,29). Reduced muscle mass and increased body fat are independ- different MHC isoforms, and that physical activity can offset these
ently and concomitantly associated with increased risk of metabolic alterations (38), future investigations should seek to determine more
disease (30), frailty (31), and mortality (32). Thus, the absence of suf- specific and hybrid fiber types. Nevertheless, the current data suggest
ficient physical activity in the face of surplus energy intake in older that chronic endurance exercise elicits a mode-specific remodeling
age increases the likelihood of unfavorable changes in body compos- of muscle fibers, such that MHC I fiber area and/or abundance is
ition (ie, reduced muscle mass and increased body fat). Furthermore, greater than OC and YC.
periods of inactivity and disuse impair muscle protein anabolism and Skeletal muscle SC, as the main source of new myonuclei, plays
accelerate the progression of sarcopenia (33). Our data demonstrate an important role in the repair and regeneration of skeletal muscle
1086 Journals of Gerontology: BIOLOGICAL SCIENCES, 2020, Vol. 75, No. 6

(39). Given that age-related MHC II fiber atrophy is accompanied young endurance-trained cohort, precludes us from determining how
by a reduction in MHC II SC, and that SC content is a strong pre- chronic exercise influences the rate of physiological decline. However,
dictor of muscle fiber size in older individuals (40,41), reductions in evidence suggests that MA experience a similar, or greater rate of
SC may impair the capacity for muscle maintenance in old age. As physiological and functional deterioration compared with their un-
such, we were surprised to find no age-related difference in fiber- trained age-matched counterparts (13,15), but the “set point” from
specific SC content between YC and OC. However, the absence of which this deterioration begins is delayed (or postponed) in MA (15).
any effect of chronic endurance exercise on SC content (per fiber) Although chronic exercise training may lower the risk of disease and
is consistent with the work of Mackey and colleagues (16) and disability in later life (46), the apparent shift to a “slow” muscle
aligned to evidence that the SC pool remains constant in response to phenotype (and reduction in MHC II fiber proportion) that we and
endurance-based training programs in humans (42). The absence of others have observed in endurance-trained MA could, paradoxically,
any difference in fiber-specific SC within or between groups could be be viewed as a hallmark of aggravated sarcopenia (47). However,

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due to a large within-group variability in the subset of samples avail- others have recently reported that long-term athletic training
able for analysis. In addition to SC content, SC activity is critical (strength or endurance) facilitates more successful reinnervation of
for muscle repair and regeneration with exercise-induced damage. denervated muscle fibers (48), which would strongly influence final
SC activity is reportedly delayed in older individuals (43), increases MHC expression. Thus, it would be prudent to examine whether the
with endurance training (42), and could therefore be maintained in observed differences in physiological function, strength, and muscle
MA and warrants further investigation. The area of the cell gov- morphology between endurance-trained MA and OC are further
erned by each myonucleus (myonuclear domain) has been reported modified in very old age (ie, >80 years). Ultimately, the inclusion of
to diminish in older age (40). Our findings provide support for an resistance-type exercise may help to preserve strength and physical
age-related reduction in myonuclear domain in MHC II, but not function in endurance-trained MA (37). To date, studies of physio-
MHC I, fibers. Interestingly, the age-related reduction in fiber-type logical function and muscle morphology in strength/power trained
myonuclear domain size was not offset in MA, analogous to find- MA are scarce.
ings from the only other investigation of myonuclei in chronically In conclusion, our findings demonstrate that endurance-trained
endurance-trained MA (16). Taken together, these data indicate that MA with an average of ~37 years of training experience display su-
larger MHC I fiber area in MA versus OC occurs independently of perior aerobic capacity, body composition, and indices of muscle
alterations in SC content or myonuclear domain, which appear rela- fiber morphology compared with age-matched untrained older in-
tively constant with ageing and chronic endurance training. Further dividuals. Irrespective of whether chronic endurance exercise offsets
to this, the age-related diminution of MHC II fiber myonuclear do- the rate of age-related physiological deterioration, or simply delays
main in MA and OC appears to precede any change in SC content. the point from which this decline begins, the novel insights shown
Impairments in muscle perfusion and the delivery of oxygen here shed light on the extent to which chronic endurance training
and nutrients to skeletal muscle from nearby capillaries may im- supports physical function and skeletal muscle health in older age,
pair muscle oxidative capacity and contribute to the development with implications for healthspan extension. Thus, the promotion of
of impaired muscle anabolism and sarcopenia. Indeed, capillary regular physical activity across the life-course, that incorporates en-
density is reduced in older age, particularly in MHC II fibers (44), durance and strength training, may offer the greatest level of pro-
thereby impairing muscle fiber perfusion. In older muscle, the C/Fi tection against the decline in physiological health and function with
and the CFPE index are reduced, and the distance between satel- advancing age.
lite cells and the nearest capillary is greater in MHC II fibers com-
pared with younger individuals (9). This may have consequences
for the responsiveness of SC to facilitate skeletal muscle repair and Supplementary Material
remodeling processes in response to contraction-induced muscle Supplementary data are available at The Journals of Gerontology,
damage. In contrast to the findings of Nederveen and coworkers Series A: Biological Sciences and Medical Sciences online.
(9), we did not observe any age-related reduction of indices of
capillarization, which may be reflective of the greater aerobic cap-
acity of our OC cohort (~10.0 mL·kg−1·minute−1 greater than that Acknowledgments
reported in Nederveen and coworkers). Nevertheless, it is clear from The authors would like to thank the research participants for their time and ef-
our results that chronic endurance exercise training leads to greater fort. The Pax7 hybridoma cells developed by Dr. A. Kawakami and the A4.951
MHC I and II fiber capillarization in MA compared with OC and, developed by Dr. H. Blau were obtained from the Developmental Studies
in some cases, YC. These findings are consistent with recent evidence Hybridoma Bank, created by the NICDHD of the NIH and maintained at
that the continuation of endurance training into older age maintains The University of Iowa, Department of Biology, Iowa City, IA 52242, USA. All
authors gave their final approval of the version of the article to be published.
skeletal muscle capillarization (45). However, recent evidence from
JM, CAG, and LB designed the study. JM and LB organized and carried out
a cross-sectional analysis of highly trained MA (55–79 years) sug-
the experiments with the assistance of BL and SB. JM, SJ, GP, and LB per-
gests that chronic endurance training does not completely prevent
formed the data analyses. JM, SJ, and LB performed the statistical analysis of
the decline in capillary density (12). Nevertheless, the greater muscle the data. JM, CAG, and LB wrote the manuscript. JM, CAG, and LB are the
fiber capillarization and MHC I fiber area observed in our cohort guarantors of this work and take responsibility for the integrity and accuracy
of endurance-trained MA may, at least partly, explain their superior of the data analysis.
aerobic fitness levels.
While our findings clearly demonstrate superior indices of
physiological function and muscle morphology in MA compared Funding
with OC, we are unable to determine whether chronic endurance This work was supported an “Exercise as Medicine” PhD studentship to
exercise offsets the trajectory of age-related physiological deteri- JM, through the College of Life and Environmental Sciences, University of
oration. The cross-sectional nature of this study, and absence of a Birmingham.
Journals of Gerontology: BIOLOGICAL SCIENCES, 2020, Vol. 75, No. 6 1087

Conflict of Interest remodeling. Physiol Rep. 2016;4(19):1–11. doi:UNSP e12987 10.14814/

phy2.12987
None reported.
19. Björkman F, Ekblom-Bak E, Ekblom Ö, Ekblom B. Validity of the re-
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