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Original article
Neuromuscular, biomechanical, and energetic adjustments following
repeated bouts of downhill running
Arash Khassetarash
a
, Gianluca Vernillo
a,b
, Renata L. Kr€
uger
a
, W. Brent Edwards
a
,
Guillaume Y. Millet
a,c,d,
*
a
Human Performance Laboratory, Faculty of Kinesiology, University of Calgary, Calgary T2N 1N4, Canada
b
Department of Biomedical Sciences for Health, University of Milan, Milan 20133, Italy
c
Inter-university Laboratory of Human Movement Biology, UJM-Saint-Etienne, Universit
e de Lyon, Saint-Etienne 42023, France
d
Institut Universitaire de France (IUF), Paris 75231, France
Received 13 January 2021; revised 9 March 2021; accepted 7 April 2021
Available online 21 June 2021
2095-2546/Ó2022 Published by Elsevier B.V. on behalf of Shanghai University of Sport. This is an open access article under the CC BY-NC-ND license.
(http://creativecommons.org/licenses/by-nc-nd/4.0/)
Abstract
Purpose: This study used downhill running as a model to investigate the repeated bout effect (RBE) on neuromuscular performance, running bio-
mechanics, and metabolic cost of running.
Methods: Ten healthy recreational male runners performed two 30-min bouts of downhill running (DR1 and DR2) at a 20% slope and 2.8 m/s
3 weeks apart. Neuromuscular fatigue, level running biomechanics during slow and fast running, and running economy parameters were recorded
immediately before and after the downhill bouts, and at 24 h, 48 h, 72 h, 96 h, and 168 h thereafter (i.e., follow-up days).
Results: An RBE was confirmed by attenuated muscle soreness and serum creatine kinase rise after DR2 compared to DR1. An RBE was also
observed in maximum voluntary contraction (MVC) force loss and voluntary activation where DR2 resulted in attenuated MVC force loss and
voluntary activation immediately after the run and during follow-up days. The downhill running protocol significantly influenced level running
biomechanics; an RBE was observed in which center of mass excursion and, therefore, lower-extremity compliance were greater during follow-
up days after DR1 compared to DR2. The observed changes in level running biomechanics did not influence the energy cost of running.
Conclusion: This study demonstrated evidence of adaptation in neural drive as well as biomechanical changes with the RBE after DR. The higher
neural drive resulted in attenuated MVC force loss after the second bout. It can be concluded that the RBE after downhill running manifests as
changes to global and central fatigue parameters and running biomechanics without substantially altering the energy cost of running.
Keywords: Cost of running; Eccentric exercise; Fatigue; Muscle damage
1. Introduction
The repeated bout effect (RBE) during unaccustomed
eccentric-biased exercises has been well documented.
1
The
RBE refers to an orchestration of neural adaptations,
2
altera-
tions to muscle mechanical properties,
3
structural remodeling
of the extracellular matrix,
4
and biochemical signaling
5
that
attenuates the extent of strength loss and delayed-onset muscle
soreness for a given exercise during subsequent bouts
(reviewed in Hyldahl et al.
1
). A majority of the literature thus
far has analyzed the RBE during single-joint tasks.
1
This is a
caveat because the neuromuscular system may distribute the
workload demands of a multijoint task differently over time.
6
Consequently, RBE observations during single-joint tasks may
not necessarily be directly translatable to dynamic multijoint
tasks such as downhill running (DR), which has become more
prevalent due to the increased interest in trail and mountain
running events.
7
DR is characterized by high levels of eccentric muscle con-
tractions in lower-limb extensor muscles, the extent of which
is dependent on the slope, duration, and speed of running.
811
Byrnes et al.
12
showed that a single bout of 30-min DR
increased serum creatine kinase (CK) and muscle soreness,
which peaked at 6 h and 42 h postexercise, respectively. Repe-
tition of the same DR protocol performed up to 6 weeks after
the initial bout substantially attenuated these measures,
12
Peer review under responsibility of Shanghai University of Sport.
* Corresponding author.
E-mail address: guillaume.millet@univ-st-etienne.fr (G.Y. Millet).
https://doi.org/10.1016/j.jshs.2021.06.001
Cite this article: Khassetarash A, Vernillo G, Kr€
uger RL, Edwards WB, Millet GY. Neuromuscular, biomechanical, and energetic adjustments following repeated
bouts of downhill running. J Sport Health Sci 2022;11:31929.
Available online at www.sciencedirect.com
Journal of Sport and Health Science 11 (2022) 319329
confirming that DR can be used as a model of exercise-induced
muscle damage to investigate RBE.
An immediate and a long-lasting reduction in maximum
voluntary contraction (MVC) force after a bout of damage-
inducing DR has been well documented.
1318
MVC force loss
is the global index of neuromuscular fatigue, the extent of
which is dictated by central or peripheral factors or by a com-
bination of the two.
19
Several studies have investigated MVC
force loss after DR using electrical nerve stimulations.
17,20,21
These studies suggested that central fatigue (originating at spi-
nal and/or supraspinal levels
19
) plays a role in MVC force loss
after DR, but persistent MVC force loss afterwards was due
predominantly to peripheral fatigue (occurring at the skeletal
muscle level
22
). An RBE in MVC force loss after prolonged
DR has been reported,
23
but the extent to which RBE may
influence the components of neuromuscular fatigue after DR
has not yet been studied. Although much of the MVC force
loss may be attributed to peripheral fatigue (i.e., disruption in
excitation-contraction mechanisms
24
), some research also sug-
gests a neural contribution to RBE.
2,25
Information about the
origin of neuromuscular fatigue may lead to a better under-
standing of the mechanisms behind the RBE
1,2
in DR.
Eccentric-biased exercise is known to increase oxygen
uptake ( _
VO2) during level running (LR) performed on subse-
quent days.
14,26
The cause(s) of elevated _
VO2after a damage-
inducing exercise are multifactorial and may be related to fac-
tors such as increased ventilation due to muscle soreness,
26
elevated resting metabolic rate,
27
or changes in running biome-
chanics.
15
If these factors were mitigated by the RBE, an atten-
uated drift in both _
VO2and energy cost of running (Cr)
28
during LR would be expected. An attenuated _
VO2drift due to
the RBE after the second DR bout has been shown.
13
Attenu-
ated ventilation due to attenuated soreness
29
after the second
bout as well as attenuated changes in running biomechanics
13
were reported as potential causes.
The Cr is closely tied to running biomechanics.
30
LR bio-
mechanics changes 24 h to 72 h after a damage-inducing bout
of DR, shifting toward an increased stride frequency and a
reduced lower-extremity range of motion.
1316
Muscle sore-
ness was introduced as a potential reason for the aforemen-
tioned changes.
13
The RBE on LR biomechanics after DR has
been investigated only in a single study,
13
which reported that
the RBE attenuated ankle and knee range-of-motion reductions
during LR when performed up to 48 h after the DR bout.
13
However, the relationship between running biomechanical var-
iables and _
VO2/Cr is more complicated; it also depends on
spatiotemporal factors, such as contact time, center of mass
excursion, and/or leg stiffness.
31
Given that these variables
have not been investigated previously, a more comprehensive
analysis of running biomechanics is needed to elucidate the
RBE on LR biomechanics after repeated DR bouts.
To summarize, the impacts of the RBE after DR on the
causes of neuromuscular fatigue (central vs. peripheral), run-
ning economy, as well as running biomechanics remain largely
unknown. Thus, this study aimed to investigate the effects of a
repeated bout of DR on acute and delayed changes in neuro-
muscular function, Cr, spatiotemporal parameters, and
external-force measurements during LR. We hypothesized that
the RBE would attenuate global, peripheral, and central indi-
ces of neuromuscular fatigue, as well as soreness in knee
extensor muscles, resulting in attenuated changes in biome-
chanics and Cr during LR.
2. Methods
2.1. Participants
Ten young healthy males (age = 25.7 §2.8 years, height =
1.76 §0.07 m, body mass = 75.6 §10.8 kg; mean §SD) par-
ticipated in this study. The participants were recruited either
through fliers around university campus or posts on social
media. The participants were recreational runners who were
already familiar with treadmill running. During the 6 months
prior to data collection, the participants had not performed pro-
longed downhill running or sustained any running-related inju-
ries. The study procedure complied with the Declaration of
Helsinki and was approved by the University of Calgary Ethics
Board (ethics number: REB 18-3093). All participants signed a
written informed-consent form. The participants were asked to
refrain from performing any strenuous exercise for 72 h before
the beginning of the protocol and during the subsequent 4
weeks of data collection.
2.2. Protocol
A schematic representation of the protocol is shown in
Fig. 1. The study was composed of 2 DR sessions separated
by 3 weeks (Fig. 1A). The participants visited the laboratory
on 13 different occasions. The 1st session (Fig. 1B) was dedi-
cated to familiarizing the participants with the experiment’s
protocol, which included neuromuscular fatigue evaluation
(NMFE) on an isometric chair (Fig. 1C), DR on an instru-
mented treadmill, and the visual perceived soreness scale. The
2nd and the 8th sessions (Fig. 1B) were dedicated to DR
bouts, hereafter called DR1 and DR2, respectively. These DR
bouts were always performed on a Monday and were exactly 3
weeks apart. Sessions 37 and 913 were performed 24 h,
48 h, 72 h, 96 h, and 168 h (POST24 to POST168) after DR1
and DR2, respectively. These sessions will hereafter be
referred to as follow-up sessions. Immediately before (PRE)
and after (POST) downhill run, follow-up sessions were dedi-
cated to blood sampling and assessing neuromuscular function,
running biomechanics, and Cr. The participant’s body mass
was measured during each visit and was used to calculate bio-
mechanical and Cr parameters.
2.3. Downhill running
Each participant performed all tests at the same time of day
(morning, afternoon, or evening §1 h) to control for within-
participant diurnal variation. DR bouts consisted of running on
an instrumented treadmill Version 2.0 (Bertec, Columbus,
OH, USA) at a 20% slope and at a constant speed of 2.8 m/s
(i.e., 10 km/h). The speed (2.8 m/s), slope (–20%), and dura-
tion of DR (30 min) were chosen based on the study done by
Martin et al.
32
320 A. Khassetarash et al.
2.4. Neuromuscular function evaluation
The NMFE was performed on the knee extensors of the right
leg using peripheral nerve stimulations delivered to the femoral
nerve trunk. The participant sat on an isometric chair with 80
˚
hip flexion and 90˚ knee flexion. The ankle was fixed to the
chair just slightly above the malleolus using a noncompliant
strap attached to a calibrated force transducer. Two NMFE trials
were performed immediately before (PRE) and 3 min after
(POST) DR1 and DR2 and at POST24 to POST168. During
each NMFE, the participant performed 3 sets of 5-s MVCs with
1-min rest in between. The 1st MVC was performed without
femoral nerve stimulation. The 2nd and 3rd MVCs were per-
formed with femoral nerve stimulation using the adapted inter-
polated twitch technique.
33
The NMFE procedure is shown in
Fig. 1C. Once a plateau was identified in the force response, 1
superimposed high-frequency doublet (Db100
sup
) was delivered
to the femoral nerve. Between 2 s and 3 s after MVC, the fol-
lowing stimulations were evoked on the relaxed muscle: high-
frequency doublet (Db100), low-frequency doublet (Db10), and
single twitch (Pt) with a 5-s interval between each. Global
fatigue was assessed by MVC loss. Db100, Db10, Pt, and the
ratio between the Db10 to Db100 (Db10:Db100, as the index of
low-frequency fatigue) were averaged between the 2 NMFE tri-
als and were used as indices of peripheral fatigue. Central
fatigue was assessed by voluntary activation (VA) level, which
was calculated using the following equation:
34
VA %ðÞ¼ 1
Db100sup Fbefore
Fbefore
Db100sup
Db100
0
@1
A100
where F
before
term is the force response just before the super-
imposed doublet was delivered.
2.5. Running biomechanics
Spatiotemporal parameters and external forces were
assessed during running at 2 different speeds (2.5 m/s (i.e.,
9km/h‒slow speed) and 3.9 m/s (i.e., 14 km/h‒fast speed))
on the same instrumented treadmill used for DR sessions for
1 min each. Ground reaction forces were recorded at 2000
Hz within the middle 20 s of each running bout to ensure
steady-state running. These bouts were performed before
(PRE) and within 10 min after (POST) DR1 and DR2 and at
POST24 to POST168. A vertical ground reaction force
threshold of 20 N was used to identify foot strike and toe-
off. Contact time, flight time, and step frequency were subse-
quently quantified from these events. The vertical impulse
was calculated from the time integral of the vertical ground
reaction force. Center of mass excursion and touchdown
velocity were calculated as follows:
COMExc:¼Z
tc
0Z
tc
0
VGRF mgðÞdtdt
U0¼1
2mZ
tc
0
VGRFðÞdt gtc
2
where dtis the time increment, COM
Exc.
is the center of mass
excursion, VGRF is the vertical component of ground reaction
force, and U
0
is the touchdown velocity. The parameters m, g,
Fig. 1. Schematic overview of the protocol (not scaled in time). (A) The 2 DR sessions were separated by 3 weeks. (B and C) The detailed description of the ses-
sions and the detailed neuromuscular function evaluations. DR = downhill running; MVC = maximal voluntary contraction; NMFE = neuromuscular function eval-
uation; NS = femoral nerve stimulation; RE = running economy.
Neuromuscular function after repeated bouts of downhill running 321
and t
c
represent total body mass, gravitational acceleration,
and contact time, respectively.
2.6. Running economy
Breath-by-breath respiratory responses were measured
using an automatic metabolic cart (Quark CPET; Cosmed,
Rome, Italy). During each visit, the resting gas exchange was
measured in a sitting position for 4 min. Participants then per-
formed bouts of 5-min LR at 2.8 m/s (i.e., 10 km/h) while the
gas exchange was measured in order to analyze running econ-
omy. These bouts were performed before (PRE) and within
15 min after (POST) DR1 and DR2, as well as at POST24 to
POST168. The last 1 min of each 5-min measurement was
used for analysis to achieve a flat, steady-state _
VO2plateau.
The following variables were considered for statistical analy-
sis: _
VO2, pulmonary ventilation ( _
VE), respiratory exchange
ratio (RER), and Cr, defined by the energy required to run
1 meter normalized to kg of body mass with a unit of J/m/kg:
Cr¼Erun Erest
168 ¢m
where E
run
is the energy equivalent in kJ/min of _
VO2during
running, E
rest
is the energy equivalent of _
VO2at rest, mis the
body mass in kg, and 168 is the distance in meters run in 1 min
at 2.8 m/s (i.e., 10 km/h).
2.7. Perceived muscle soreness
Borg’s category ratio 10 (CR10) pain scale
35
was used to
assess perceived muscle soreness (SOR). Participants reported
their perceived SOR in the quadriceps muscle group at the
static squatting position. Participants were instructed always to
start by looking at the verbal expressions on the CR10 scale
and then choose a number between 0 (nothing at all) and 10
(extremely strong) or higher than 10 (absolute maximum).
2.8. Blood sampling
Blood sampling was performed before (PRE) and within
half an hour after (POST) DR1 and DR2 as well as at POST48
and POST72. Approximately 4 mL of blood were drawn from
the antecubital vein into serum vacutainers (red stoppers).
After sitting for at least 30 min at room temperature for clot
formation, the blood samples were centrifuged for 15 min at
3000 £gat 48C to obtain blood serum, which was stored for
24 weeks at 208C. Blood serum samples were then ana-
lyzed (Cobas 8000, Roche Diagnostics, Basel, Switzerland)
for CK concentration.
2.9. Statistical analysis
All variables were checked for normality and the results
were reported as mean §SD. Neuromuscular fatigue parame-
ters, running economy parameters, running biomechanics
parameters, and muscle soreness were considered as dependent
variables. All dependent variables were normalized to baseline
(baseline was the magnitude of the dependent parameter of
interest at PRE) except SOR, CK, VA, and Db10:Db100. To
assess the differences between PRE and POST DR (fatigue
effect) as well as muscle damage (PRE, POST24 to
POST168), a longitudinal analysis (bout £time) was per-
formed using generalized estimating equations (GEE; General-
ized Linear Model Procedure). If significant main effects or
interactions were observed, pairwise comparisons were
adjusted for multiple comparisons using the Bonferroni correc-
tion. All statistical analyses were performed using IBM SPSS
Statistics (Version 26.0.0; IBM Corp., Armonk, NY, USA)
with the criterion a-level set to 0.05.
3. Results
Numerical values for all the variables investigated at all
time points, along with their corresponding statistics, are pre-
sented in Supplementary Table 1.
3.1. Blood CK and SOR
Blood samples were taken from a subset of participants
(n= 6). A significant time £bout interaction (x
2
(3) = 15.1,
p= 0.002) was observed for CK (Fig. 2A). After DR1, the
blood CK increased from 232 U/L at PRE to 368 U/L, 522 U/L,
and 438 U/L (all p<0.011) at POST, POST48, and POST96,
respectively. After DR2, CK did not significantly increase (all
p>0.078).
A significant bout £time interaction (x
2
(6) = 126.1, p<
0.001) was observed for SOR (Fig. 2B). After DR1, SOR
increased by 2.73 units (p<0.001) and remained elevated
Fig. 2. (A) Serum CK and (B) perceived muscle soreness at different measurement points. Values are means §SD. * p<0.05, significent difference between
bouts;
#
p<0.05, significent difference from PRE in DR1;
y
p<0.05, significent difference from PRE in DR2. CK = creatine kinase; DR1= downhill running 1;
DR2 = downhill running 2; PRE = before downhill run; SOR = perceived muscle soreness.
322 A. Khassetarash et al.
until POST96 (all p<0.05) and returned to the baseline at
POST168. After DR2, SOR increased by 0.72 units (p<
0.001), remained elevated at POST24 (p<0.011), and
returned to baseline at POST48. Furthermore, SOR was higher
after DR1 at POST, POST24, POST48, and POST72 as com-
pared to DR2 (all p<0.027).
3.2. Neuromuscular function
A significant bout £time interaction (x
2
(6) = 54.1, p<
0.001) was observed for MVC (Fig. 3A) (Table 1). After
DR1, MVC dropped by 16.5% (p<0.001) at POST and
remained lower than baseline until POST48 (all p<0.004).
After DR2, MVC dropped by 8.4% (p<0.001) and recovered
by POST24 (p= 0.083). Reductions in MVC during follow-up
days of DR1 at POST, POST48, and POST96 were signifi-
cantly greater than those of DR2 (all p<0.032).
The results for the peripheral fatigue variables (Fig. 3B‒D)
(Table 1) were similar to each other, i.e., a significant time
effect, but no significant bout £time interaction was observed.
On average, Db100, Db10:Db100, and Pt dropped by 14.9%,
26%, and 24% (all p<0.001) at POST and returned to base-
line by POST48.
A significant bout £time interaction (x
2
(6) = 285.2, p<
0.001) was observed for VA (Fig. 3E) (Table 1). After DR1,
VA dropped by 8.6% (p<0.001) at POST and returned to
baseline at POST24 (p= 0.366). After DR2, VA did not
change either at POST or during recovery days (all p>0.242).
VA at POST DR1 was 5.8% (p<0.001) lower than that at
POST DR2. Furthermore, VA after DR1 was lower than after
DR2 from POST24 to POST72 (all p<0.015) and at POST
168 (p= 0.025).
3.3. Running biomechanics
A significant bout £time interaction (x
2
(6) = 86.0, p<
0.001) was observed for contact time at fast speed (Fig. 4A)
(Table 1). After DR1, this variable increased by 2.5% (p<
0.001) at POST and then reduced by 3.1% (p<0.001) at
POST24 and remained lower than baseline (all p<0.018)
until POST168 (p= 0.112). After DR2, contact time decreased
by 2.7% at POST (p<0.001) and returned to baseline at
POST24 (p= 0.317). Furthermore, changes in contact time at
fast speeds were larger during DR1 than DR2 at POST24 and
POST72 (all p<0.02).
A significant bout £time interaction (x
2
(6) = 22.9,
p= 0.001) was observed for flight time at slow speed (Fig.
4B) (Table 1). Only after DR1 at POST24 was the flight time
smaller than PRE (p= 0.029). Furthermore, the change in flight
time was smaller for DR1 than DR2 at POST24 (p= 0.019).
A significant bout £time interaction was not observed for
step frequency at slow or fast speeds (Table 1). A significant
time effect was observed for step frequency at both slow and
fast speeds (Fig. 4C) (Table 1). Step frequency at POST
increased by 2.1% and 1.6% (all p<0.009) for slow and fast
speeds, respectively, and returned to baseline at POST72.
A significant bout £time interaction (x
2
(6) = 33.3, p<
0.001) was observed for vertical impulse at slow speed (Fig. 4D)
Fig. 3. (A) Percent changes in MVC; (B) Db100; (C) Db10:Db100; (D) Pt; and (E) VA normalized to PRE (except for VA and Db10:Db100). Values are
means §SD. * p<0.05, significant difference between bouts;
#
p<0.05, significant difference from PRE in DR1;
y
p<0.05, significant difference from PRE
in DR2. Db100 = maximum force evoked by a 100 Hz doublet; Db10:Db100 = maximum force evoked by a 10 Hz doublet divided by Db100; DR1 = downhill
running 1; DR2 = downhill running 2; MVC = maximum voluntary contraction; Pt = potentiated twitch force evoked by single twitch; PRE = before downhill
run; VA = voluntary activation, calculated by the twitch interpolation technique.
Neuromuscular function after repeated bouts of downhill running 323
(Table 1). On average, vertical impulse after DR1 was reduced
by 2.4% at POST (p<0.001) and remained lower than baseline
until POST48 (all p<0.003) and returned to baseline at
POST72. Vertical impulse after DR2 was reduced by 2.8% (p<
0.001) and returned to baseline at POST48.
A significant bout £time interaction (x
2
(6) = 34.4, p<
0.001) was observed for center of mass excursion at slow
speed (Fig. 4E) (Table 1). On average, this variable did not
change after DR1 (all p>0.055), whereas it was reduced by
10.4% (p= 0.001) after DR2 at POST24. The change in this
variable, when compared to PRE, was larger for DR2 than
DR1 at all measurement points (all p<0.046) with the excep-
tion of POST72 (p= 0.099). A significant bout £time interac-
tion (x
2
(6) = 40.4, p<0.001) was observed for center of mass
Table 1
Baseline values (PRE downhill run), Wald x
2
, and pvalue for the NMF, running economy, and running biomechanics variables.
Variable Bout Absolute at PRE Time Bout Time £bout
Wald x
2
pWald x
2
pWald x
2
p
NMF
MVC (N) DR1 693.0 §116.5 89.4 <0.001 5.8 0.016 54.1 <0.001
DR2 667.5 §120.5
VA (%) DR1 91.0 §4.3 42.2 <0.001 17.9 <0.001 285.2 <0.001
DR2 92.9 §3.7
Db100 (N) DR1 294.2 §48.2 234.1 <0.001 0.0 0.85 37.2 <0.001
DR2 302.2 §57.4
Db10:Db100 DR1 1.06 §0.04 165.2 <0.001 0.2 0.628 2.2 0.895
DR2 1.06 §0.06
Pt (N) DR1 202.8 §40.9 510.7 <0.001 0.3 0.60 9.5 0.145
DR2 204.5 §45.9
SOR DR1 0 77.5 <0.001 18.4 <0.001 126.1 <0.001
DR2 0
Running economy
_
VO2(mL/min/kg) DR1 39.0 §3.0 9.9 0.13 0.3 0.59 78.4 <0.001
DR2 39.5 §2.9
Cr (J/m/kg) DR1 4.1 §0.4 17.2 0.009 0.1 0.75 8.8 0.187
DR2 4.2 §0.5
_
VE (mL/min) DR1 82.4 §11.3 15.9 0.014 0.6 0.45 349.0 <0.001
DR2 81.2 §12.1
RER DR1 0.91 §0.06 137.9 <0.001 0.01 0.99 59.0 <0.001
DR2 0.90 §0.06
Running biomechanics
Contact time (slow; s) DR1 0.30 §0.03 24.1 <0.001 0.1 0.80 5.2 0.516
DR2 0.30 §0.03
Contact time (fast; s) DR1 0.22 §0.00 57.4 <0.001 1.7 0.19 86.0 <0.001
DR2 0.22 §0.02
Flight time (slow; s) DR1 0.076 §0.03 16.5 0.011 1.4 0.24 22.9 0.001
DR2 0.077 §0.03
Flight time (fast; s) DR1 0.12 §0.02 21.3 0.002 0.8 0.38 3.4 0.754
DR2 0.12 §0.02
Step frequency (slow; Hz) DR1 2.69 §0.09 140.9 <0.001 0.01 0.85 7.4 0.288
DR2 2.66 §0.11
Step frequency (fast; Hz) DR1 2.88 §0.12 73.4 <0.001 0.2 0.68 3.9 0.685
DR2 2.86 §0.13
Vertical impulse (slow; N.s) DR1 278.9 §44.2 47.5 <0.001 0.01 0.96 33.3 <0.001
DR2 282.2 §44.0
Vertical impulse (fast; N.s) DR1 258.8 §40.3 57.7 <0.001 0.8 0.36 3.8 0.710
260.0 §39.0
COM
EXC.
(slow; cm) DR1 5.8 §0.7 164.6 <0.001 12.5 <0.001 34.4 <0.001
6.3 §1.1
COM
EXC.
(fas; cm) DR1 5.7 §1.4 66.3 <0.001 0.01 0.95 40.4 <0.001
DR2 5.6 §1.3
Touchdown velocity (slow; m/s) DR1 0.37 §0.13 61.3 <0.001 1.4 0.25 2.5 0.867
DR2 0.42 §0.12
Touchdown velocity (fast; m/s) DR1 0.60 §0.10 58.9 <0.001 0.3 0.58 1.9 0.929
DR2 0.63 §0.95
Notes: The baseline values for all variables investigated were not different between DR1 and DR2. The absolute values are presented as mean §SD.
Abbreviations: COM
EXC.
= center of mass excursion; Cr = cost of running; Db100 = maximum force evoked by 100 Hz doublet; Db10:Db100 = maximum force
evoked by a 10 Hz doublet divided by Db100; MVC = maximum voluntary contraction; NMF = neuromuscular function; Pt = potentiated twitch force evoked by
single twitch; PRE = before downhill run; RER = respiratory exchange ratio; SOR = perceived muscle soreness; VA = voluntary activation; _
VE = pulmonary venti-
lation; _
VO2= oxygen uptake.
324 A. Khassetarash et al.
excursion at fast speed (Fig. 4E) (Table 1). After DR1, this
variable reduced by 9.2% (p<0.001) at POST24 and returned
to baseline at POST96. After DR2, this variable reduced by
6.9% (p= 0.007) at POST24 and returned to baseline at
POST72. Furthermore, only at POST24 was the change in cen-
ter of mass excursion higher for DR1 than DR2 (p= 0.039).
A significant bout £time interaction was not observed for
touchdown velocity at either slow or fast speeds (Table 1). A sig-
nificant time effect was observed for touchdown velocity at both
slow and fast speeds (Fig. 4F) (Table 1). On average, touchdown
velocity was reduced by 11.9% and 6.6% (all p<0.001) for slow
and fast speeds at POST, respectively, and returned to baseline at
POST48 for slow speed and at POST24 for fast speed.
3.4. Running economy parameters
A significant bout £time interaction (x
2
(6) = 78.4, p<
0.001) was observed for _
VO2(Fig. 5A) (Table 1). After DR1,
_
VO2at POST24 was 2.9% higher than baseline, while there
was no difference compared to PRE on other days (all p>
0.139) or after DR2 (all p>0.378). Furthermore, the increase
in _
VO2after DR1 was 2.4% (p= 0.019) higher than that after
DR2 at POST48.
A significant bout £time interaction (x
2
(6) = 8.8,
p= 0.187) was not observed for Cr (Fig. 5B) (Table 1).
Although the time effect (x
2
(6) = 17.195, p= 0.009) was sig-
nificant for Cr, pairwise comparisons did not show any signifi-
cant differences from the baseline (all p>0.146). Further
metabolic variables are shown in Fig. 5 and Table 1.
4. Discussion
This study investigated the effects of repeated bouts of DR
on neuromuscular function and their possible translation to LR
biomechanics and Cr. It was hypothesized that performing the
second DR bout would result in attenuated global, peripheral,
and central fatigue and SOR and, thereby, attenuated changes
in running biomechanics and Cr. The presence of an RBE was
confirmed by attenuated CK, force loss, and perceived sore-
ness after DR2. An RBE was also present for select LR bio-
mechanical variables, but, contrary to our hypothesis, this did
not translate to an RBE for Cr.
Fig. 4. Mean values of percent changes in biomechanical parameters: (A) CT; (B) FT; (C) SF; (D) VI; (E) COM
EXC.
; and (F) U0 normalized to PRE. For clarity,
standard deviations were not presented. Solid lines (DR1) and dashed lines (DR2). Slow running (2.5 m/s) results are in black lines and fast running (3.9 m/s)
results are in gray lines in all panels. * p<0.05, significant difference between bouts;
#
p<0.05, significant difference from PRE in DR1;
y
p<0.05, significant
difference from PRE in DR2. Black markers denote significance in slow running, and gray markers denote significance in fast running. COM
EXC.
= center of mass
excursion; CT = contact time; DR1 = downhill running 1; DR2 = downhill running 2; FT = flight time; PRE = before downhill run; SF = step frequency; U0 = touch-
down velocity; VI = vertical impulse.
Neuromuscular function after repeated bouts of downhill running 325
4.1. Fatigue and RBE
Immediately after DR1, there was a significant reduction in
MVC, the magnitude of which (16.5%) was consistent with
the literature.
13,14,18,21,23
This MVC force loss was partly due
to peripheral fatigue, particularly low-frequency fatigue as
demonstrated by a decrease in Db10:Db100. Low-frequency
fatigue likely occurred due to impairments in 1 or more mech-
anisms involved in excitation-contraction coupling,
36
such as
decoupling at the T-tubule-sarcoplasmic reticulum interface
and/or a decreased Ca
2+
release from the sarcoplasmic reticu-
lum.
36
Central fatigue was also present, as indicated by a
decrease in VA. The deficit in central activation after DR was
likely due to several yet unknown factors, such as changes in
the intrinsic properties of the motoneuron pool
19
or
disfacilitation of Ia afferents.
37
The observed reductions in the
Db10:Db100 ratio and the VA are in line with those found in
the previous literature.
21,32
Immediately after DR2, MVC also decreased (8.4%); how-
ever, the magnitude of MVC force loss was lower at DR2 than
DR1, which is in line with a previous study.
23
Conversely,
Chen et al.
13
did not find any RBE for MVC after DR;
although the time between their 2 DR sessions was only 5
days, whereas in the current study, it was 3 weeks. Possible
mechanisms contributing to the RBE are increased muscle-ten-
don stiffness via reduced myotendinous junction displace-
ment
38
and extracellular remodeling.
4
Increased stiffness may
prevent muscle fibers from over-straining, which results in less
muscle damage and less reduction in force-production capac-
ity.
38
Furthermore, a recent review suggested that the strength-
ening of the extracellular matrix starts more than 2 days after
eccentric exercise.
1
It may, therefore, be possible that a 5-day
period was not long enough to elicit an RBE in the overall
muscle strength loss. The observed RBE for MVC was accom-
panied only by an RBE for VA. Therefore, the results of the
current study suggest that the participants were less fatigued
after DR2 than after DR1. The RBE may be explained partly
by a modification in voluntary drive. Possible mechanisms
include: (1) decruitment of faster motor units and preferential
recruitment of additional slower motor units;
39
(2) enhanced
synchronization of motor units;
39
(3) attenuated level of supra-
spinal fatigue;
2
and (4) less disfacilitation of Ia afferents.
37
In the present study, running biomechanics were substan-
tially affected immediately after both DR sessions. A signifi-
cant increase in step frequency and a concomitant reduction in
flight time during slow running suggested a transition to a
“shuffling” type of running gait. This was further confirmed by
reductions in touchdown velocity and vertical impulse. Such a
reduction in touchdown velocity indicates a lower kinetic
energy at touchdown, which would result in lower breaking
impulse and energy absorption (e.g., eccentric work). This
transition may have occurred to minimize the pain and reduce
the eccentric workload on already damaged knee extensor
muscles. The results were somewhat similar during fast run-
ning, except that contact time also increased immediately after
both DR sessions. This suggests a transition to a more
“grounded running” style. An RBE was evident for MVC and
VA, but most biomechanical variables did not show any RBE
at POST. One explanation is that the amount of muscle force
needed for submaximal running is only a fraction of the maxi-
mum capacity of the locomotor muscles.
40
Therefore, the
reduction in maximal force may not substantially translate into
changes in running biomechanics. Center of mass excursion
during slow running was the only biomechanical variable that
showed an RBE at POST. An increased center of mass excur-
sion could potentially lead to an increased Cr
31
due to the
greater mechanical work required to move vertically; however,
this was not observed. _
VO2and Cr depend on numerous varia-
bles, such as muscle-tendon unit stiffness,
41
muscular
strength,
42
and running biomechanics.
31
Running biomechani-
cal parameters, such as leg stiffness,
41
step length,
43
and con-
tact time
44
have been shown to affect _
VO2. In general, it
Fig. 5. (A) Percent changes in _
VO2; (B) Cr; and (C) _
VE normalized to PRE.
Values are means §SD. *p<0.05, significant difference between bouts;
#
p<0.05, significant difference from PRE in DR1;
y
p<0.05, significant
difference from PRE in DR2. Cr = cost of running; DR1 = downhill running 1;
DR2 = downhill running 2; PRE = before downhill run; _
VE = pulmonary venti-
lation; _
VO2= oxygen consumption.
326 A. Khassetarash et al.
appears that changes in running biomechanics were not of a
magnitude and/or direction to affect Cr substantially.
4.2. RBE during follow-up days
In agreement with the previous studies,
12,13,16
we observed
an attenuated response in the primary markers of muscle dam-
age following DR2. Specifically, CK and ratings of muscle
soreness were lower, whereas the recovery of maximal force-
generating capacity was accelerated. Furthermore, higher VA
following DR2 lends support to the notion that the RBE is
mediated by a neural drive adaptation.
45
This might be a con-
sequence of the altered recruitment strategy during the
repeated activity that reduced the magnitude of damage.
2
In-
terestingly, no RBE was observed for Db10:Db100. This is
surprising because low-frequency fatigue is a prominent char-
acteristic of exercises involving lengthening contractions of
the active muscles, such as eccentric-biased DR.
32
Accord-
ingly, an attenuated response for Db10:Db100 was expected
following DR2, in agreement with the notion that the RBE is
mediated by mechanical adaptation.
45
However, methodologi-
cal limitations in the assessment of Db10:Db100 using paired
stimulations vs. trains of stimulation can be called upon as a
potential explanation. Although the 2 methods were found to
be correlated after DR,
46
the extent of low-frequency fatigue is
underestimated using paired stimulations because of a much
lower release of Ca
2+
from the sarcoplasmic reticulum follow-
ing a pair vs. a train of stimuli at high frequencies.
47
Biomechanics of LR at slow and fast speeds remained
altered up to 72 h after both DR1 and DR2. The paramount
alteration during both slow and fast running was elevated step
frequency. Increased step frequency after DR has been
reported previously,
1315,48
and was thought to be related to a
reduction in lower extremity joint range of motion
14,15
and,
probably, stiffer joints. Increased SOR might have caused
higher discharge of Type III and IV afferents;
29
therefore, it
can be speculated that reduction in ranges of motion may help
reduce strain within the muscle and, hence, maintain SOR at a
tolerable level. For example, Hamill et al.
49
reported a reduc-
tion in maximum knee flexion 2 and 5 days after DR and sug-
gested that this alteration may help reduce stretching of the
already damaged quadriceps muscles. However, if this deduc-
tion were true, then the effect of muscle damage on step fre-
quency should be greater when a larger range of motion is
required, such as in fast running vs. slow running. Although 1
study showed more altered step frequency in fast running vs.
slow running,
14
our results did not demonstrate any noticeable
speed effect for step frequency. The role of SOR in alterations
of running biomechanics, however, cannot be ruled out by this
result. Considering the complexity of the running task, altered
step frequency may have been achieved by changes in other
spatiotemporal variables, such as contact time or flight time. In
fact, our results show different mechanisms involved in slow
and fast running. Unlike slow running, elevated step frequency
was accomplished in fast running by a reduction in contact
time and center of mass excursion, which suggests a stiffer leg
compared to baseline; these results are in line with previous
observations.
14,15
A stiffer whole-leg implies a smaller lower-
extremity joint excursion,
50
thus reducing the mechanical
strain within the muscle-tendon units, possibly to cope with
the sensation of soreness. Chen et al.
13
also reported an RBE
on step frequency, showing that only the first DR bout resulted
in elevated step frequency. However, our results indicated no
RBE for this parameter. Considering the argument mentioned
with respect to the relationship between SOR and LR biome-
chanics, it is possible that the work done by Chen et al.
13
and
our protocol (and/or participants’ fitness levels) resulted in dif-
ferent influences of the repeated bout on SOR and, conse-
quently, on step frequency.
Finally, the observed differences in both neuromuscular and
running biomechanics variables did not substantially translate
into metabolic changes during LR. The repeated bout affected
only _
VO2at POST48, which is in line with a previous observa-
tion.
13
However, Cr did not show any time effect or RBE. An
increased resting _
VO2has been demonstrated following eccen-
tric-biased exercise;
27
however, the change in resting _
VO2was
taken into account when assessing Cr in the present study. One
explanation for the observed consistency in Cr is that the
amount of damage within the first DR bout was not enough to
elicit meaningful differences in Cr when compared to Chen
et al.
14
Moreover, it is also possible that the participants opti-
mized their running biomechanics and/or muscle recruitment
to maintain Cr. Future research could use electromyography
recording to help assess such speculations.
4.3. Limitations
The same constant speed was chosen for all the participants
during DR. This is an intrinsic limitation to prescribing an
appropriate intensity to induce damage during DR. An appro-
priate constant speed during DR cannot be determined solely
by maximal oxygen uptake, given that DR is more mechani-
cally demanding than aerobically demanding; this emphasizes
the importance of factors such as maximal knee extensor
strength and muscle-tendon unit stiffness.
51
Furthermore, the
sample size for CK analysis was only a subset of participants.
Still, despite the small sample size, the DR protocol induced
great enough damage that it was statistically significant.
Finally, the properties of the running surface can potentially
affect running biomechanics. For example, running on grass
may result in longer step lengths and contact times compared
to running on a track,
52
which would influence leg-stiffness
measurements.
53
In the current study, participants performed
all running trials on a Bertec instrumented treadmill with an
appreciably stiff surface. Considering that the treadmill surface
was the same for all biomechanical measurements, we do
not feel that a different surface would affect the observed
differences between experimental conditions or, in turn, our
interpretation of the study findings.
5. Conclusion
The results of this study suggest that the adaptation in the
neural drive is an important contributor to the observed
repeated bout effect in knee extensors’ MVC force loss
Neuromuscular function after repeated bouts of downhill running 327
immediately and up to 72 h after repeated bouts of downhill
running. The acute and delayed reductions in MVC and run-
ning biomechanics did not substantially translate to metabolic
parameters during level running. It can be concluded that the
RBE after downhill running manifests as changes to global
and central fatigue parameters and running biomechanics with-
out substantially altering the Cr.
Acknowledgments
We thank the dedicated group of participants for their time
and effort. We would also like to thank Michael Baggaley,
Michael Esposito, Colin Lavigne, Stacy Lobos, Dr. Rogerio N.
Soares, and Dr. Saied Jalal Aboodarda for their help with data
collection.
Authors’ contributions
AK collected, analyzed, and interpreted the data and
drafted, edited, and revised the manuscript; GV and RLK col-
lected and interpreted the data, and drafted, edited, and revised
the manuscript; WBE and GYM designed the research, inter-
preted the data, and edited and revised the manuscript. All
authors have read and approved the final version of the manu-
script, and agree with the order of presentation of the authors.
Competing interests
The authors declare that they have no competing interests.
Supplementary materials
Supplementary materials associated with this article can be
found in the online version at doi:10.1016/j.jshs.2021.06.001.
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