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Effects of different inspiratory 
muscle warm‑up loads 
on mechanical, physiological 
and muscle oxygenation responses 
during high‑intensity running 
and recovery
Anita B. Marostegan1*, Claudio A. Gobatto1, Felipe M. Rasteiro1, Charlini S. Hartz2, 
Marlene A. Moreno2 & Fúlvia B. Manchado‑Gobatto1
Inspiratory muscle warm‑up (IMW) has been used as a resource to enhance exercises and sports 
performance. However, there is a lack of studies in the literature addressing the effects of different 
IMW loads (especially in combination with a shorter and applicable protocol) on high‑intensity running 
and recovery phase. Thus, this study aimed to investigate the effects of three different IMW loads 
using a shorter protocol on mechanical, physiological and muscle oxygenation responses during 
and after high‑intensity running exercise. Sixteen physically active men, randomly performed four 
trials 30 s all‑out run, preceded by the shorter IMW protocol (2 × 15 breaths with a 1‑min rest interval 
between sets, accomplished 2 min before the 30 s all‑out run). Here, three IMW load conditions 
were used: 15%, 40%, and 60% of maximal inspiratory pressure (MIP), plus a control session (CON) 
without the IMW. The force, velocity and running power were measured (1000 Hz). Two near‑infrared 
spectroscopy (NIRS) devices measured (10 Hz) the muscle’s oxygenation responses in biceps brachii 
(BB) and vastus lateralis (VL). Additionally, heart rate (HR) and blood lactate ([Lac]) were also 
monitored. IMW loads applied with a shorter protocol promoted a significant increase in mean and 
minimum running power as well as in peak and minimum force compared to CON. In addition, specific 
IMW loads led to higher values of peak power, mean velocity (60% of MIP) and mean force (40 and 60% 
of MIP) in relation to CON. Physiological responses (HR and muscles oxygenation) were not modified 
by any IMW during exercise, as well as HR and [Lac] in the recovery phase. On the other hand, 40% 
of MIP presented a higher tissue saturation index (TSI) for BB during recovery phase. In conclusion, 
the use of different loads of IMW may improve the performance of a physically active individual in a 
30 s all‑out run, as verified by the increased peak, mean and minimum mechanical values, but not in 
performance assessed second by second. In addition, 40% of the MIP improves TSI of the BB during 
the recovery phase, which can indicate greater availability of  O2 for lactate clearance.
High-intensity exercise such as tethered sprints is often applied in training  programs1–3. Considering the sig-
nificance of the running power in sports and training programs, investigations have been conducted to improve 
the consistency of this parameter by making it measurable using a tethered system capable of monitoring the 
force, the velocity, and consequently the running  power2,4–6. Furthermore, by applying a high-intensity exer-
cise test, especially with duration around 30 s (i.e., 30 s all-out run), it is possible to assess the peak, mean and 
minimum power, as well as the fatigue  index7–9, which contribute to training programs applied to athletes and 
active participants. The 30 s all-out run is characterized by a predominantly anaerobic stimulus that increases 
the physiological responses, such as heart rate and blood lactate  production7,8,10, by locomotor muscles (more 
OPEN
1Laboratory of Applied Sport Physiology, School of Applied Sciences, University of Campinas, Limeira, São Paulo, 
Brazil. 2Postgraduate Program in Human Movement Sciences, Methodist University of Piracicaba, Piracicaba, Sao 
Paulo, Brazil. *email: 
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active muscles). Despite the significance of the anaerobic metabolism, the aerobic metabolism also plays an 
important role during high-intensity exercise, as observed in the Wingate  test11–13 and the 30 s all-out tethered 
 run7. In addition, the recovery process is essential to reestablish the energy stores, with significant participation 
of the aerobic metabolism during and after high-intensity exercise 10,14, acting as an ally in the performance of 
subsequent intense  efforts15. Recently, our group observed that during an all-out 30 s running test the biceps 
brachii (less active muscle) showed increased deoxygenation and a quick adjustment of post-effort oxygen levels 
compared to the vastus  lateralis10. These findings reinforce the tissue-dependent response, evidencing that the 
organism adapts to a stressful stimulus in an integrative way.
Considering the integrative biology during physical  effort16 in high-intensity exercise, it is known that the 
oxygenation is directed to areas with higher demand, promoting vasoconstriction in less active muscles and vaso-
dilation in more active muscles. In this sense, the accumulation of metabolites (e.g., blood lactate and H + ions) in 
the fatigued inspiratory muscles (IM) can initiate the inspiratory muscle metaboreflex, which triggers sympathetic 
nerve activity that promotes adrenergic vasoconstriction, provoking competition with locomotor muscles for 
 oxygenation17–20, and consequently impairing the exercise performance. In line with this, it is reported that IM 
plays a significant role during exercise and  recovery19–22. Therefore, some studies have focused on IM training, 
aiming at reducing the IM fatigue in order to maximize performance and oxygenation  redistributions23–25. Moreo-
ver, the warm-up or pre-activation of inspiratory muscles (IMW) using a portable device has been suggested to 
prepare this specific musculature before exercise, and consequently improve the physiological responses during 
 effort26–28, as well as sports  performance29–32. Positive effects of IMW were observed during high-intensity, short-
duration exercises, with increased power in the Wingate  test33,34. Furthermore, better oxygenation distributions 
in more active  muscles35 and decreased lactate accumulation in athletes after intermittent  running36 were also 
observed. However, the available literature lacks studies on the effects of IMW on more and less active muscles, 
which are essential for oxygen uptake and metabolite removal,  respectively10,37.
Different IMW protocols (i.e., number of sets, repetitions and time between sets) and loads (characterized 
by airflow restriction in the inspiration phase) ranging from 5 to 80% of maximal inspiratory pressure (MIP) 
have been  proposed29,31,32,38–41. Nonetheless, most studies that applied this strategy used efforts based on indi-
vidual MIP, generally composed of 2 sets of 30 repetitions at 40% of MIP. This protocol has been suggested to 
improve exercise performance through the preparation of the inspiratory muscles, to minimize the effects of 
inspiratory muscle metaboreflex, reducing fatigue and improving oxygen delivery between the locomotor and 
respiratory  muscles29,31,34,40. The application of higher inspiratory loads such as 60–80% of  MIP38,39 to improve 
subsequent results is still controversial and lower loads such as 5–15% of MIP are commonly applied as a placebo 
 condition27,31,35,36,39. Concerning the number of repetitions, recent investigations have suggested a shorter IMW 
protocol consisting of 2 sets of 15 inspiratory efforts in order to minimize the time spent during warm-up and 
improve its application in a sports/physical training  environment32,38,39. However, there is a lack of knowledge 
about the effects of different inspiratory loads of IMW on the mechanical and physiological performance in 
high-intensity and short-duration running, especially when performing such a short inspiratory protocol.
Therefore, this study aims to investigate the effects of different IMW loads (15%, 40% and 60% of MIP) using 
a shorter protocol (2 sets of 15 repetitions with a 1-min rest interval between them) on the mechanical (i.e., 
power, force and velocity), physiological (i.e., heart rate and blood lactate) and muscle oxygenation responses 
(in vastus lateralis and biceps brachii, considered the more and less active muscles, respectively) during high-
intensity running and passive recovery. Our hypothesis is that IMW at 40% of MIP will minimize IM fatigue, 
allowing redirect the oxygenation to more active muscles with higher percentages of tissue saturation index (TSI), 
consequently promoting a positive impact on the mechanical responses during the 30 s all-out run (especially 
on mean running power and fatigue index), as well as contributing to a better post-effort blood lactate removal 
in relation to other loads. In addition, as studies are controversial and there are gaps about the IMW effects on 
high-intensity running, compared with the responses already observed in the literature in different populations 
and types of exercise it is estimated that the load with 15% of MIP has a placebo effect and is equal to the control 
session, while the 60% load does not improve performance, as it is considered a heavy load that will fatigue the 
respiratory muscles.
Materials and methods
Ethics approval. 
All procedures were approved by the local Research Ethics Committee of the School of 
Medical Sciences of the State University of Campinas (protocol number: 99783318.4.0000.5404) and were in 
accordance with the ethical recommendations in the Declaration of Helsinki. Participants were only evaluated 
after having received information about the experimental procedures and risks and signing an informed consent 
form.
Participants. 
Sixteen physically active young men (local sports team players and street running partici-
pants) were evaluated (23 ± 1 years old, 73.2 ± 2.0 kg; 177.4 ± 1.9 cm; 9.0 ± 0.5% body fat, IPAQ at 4539 ± 942 
metabolic equivalent-min/week; MIP at 145.6 ± 9.9  cmH2O, peak of global strength index (S-Index) at 139 ± 3 
and mean S-Index at 123 ± 4,  cmH2O). The analysis with G*Power software showed that a sample size of at least 
12 individuals would be necessary to obtain a power of 80% with a significance level α = 5%, based on previously 
published  data10. The participants were invited to answer questionnaires about their levels of physical activity 
(International Physical Activity Questionnaire—IPAQ), sports practice and health history. Only participants 
that presented a minimum score to classify them as ‘physically active’ were included in the  study42. Individuals 
that reported metabolic, cardiovascular, respiratory or orthopedic disease were excluded from this research.
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Experimental approach to the problem. 
The experimental design involved six visits to the laboratory 
under similar conditions and at identical daytime (± 60 min), separated by 48–72 h (Fig. 1). On the first day, 
after signing the informed consent form and completing the questionnaires, anthropometric and body composi-
tion measurements were performed. In the second visit, MIP and S-Index were determined 1 h apart to prevent 
inspiratory fatigue. On the same day, the participants were familiarized with the inspiratory muscle warm-up 
protocol and the non-motorized treadmill (NMT), when they were asked to perform five sprints of 10 s. In the 
next four visits to the laboratory, all participants were submitted to high-intensity tethered exercise (30 s all-out 
run), preceded by different IMW loads protocol (15%, 40% and 60% of MIP plus a control (CON) session with-
out the IMW protocol). These sessions were randomly performed by the individuals under the IMW protocols 
used. Upon arrival at the laboratory, the participants were equipped with near-infrared spectroscopy (NIRS) 
devices attached to the biceps brachii (BB) and vastus lateralis (VL) muscles, and a heart rate monitor (HR) for 
data acquisition throughout the session. The participants remained at rest for 3 min for the determination of 
baseline values (BL), including blood lactate concentration ([Lac]) at rest. Then, they were asked to warm-up on 
a motorized treadmill for 5 min (7 km/h and 1% inclination) and rest for another 5 min. Subsequently, the IMW 
protocol was performed. Two minutes after, the individuals were submitted to 30 s high-intensity tethered run-
ning for data acquisition (i.e., force, velocity and running power). Immediately after the test (T0), blood samples 
were collected every 2 min up to 18 min of passive recovery (T2‒T18). In all sessions, the participants were 
instructed to have a light meal, not to consume alcohol/caffeine and not to practice moderate-intense exercise 
24 h before the tests. The procedures were performed in a controlled and isolated laboratory environment, and 
the participants did not receive any information about each intervention.
Figure 1.  (A) Experimental protocol illustrating the procedures performed on each day during the whole 
study period. (B) Timeline of the protocol sessions. Physiological responses were monitored throughout the 
exercise and the 18 min of passive recovery. Near-infrared spectroscopy devices (NIRS, represented by the red 
rectangles) provided the biceps brachii and vastus lateralis oxygenation data. S-Index—global strength index; 
CON—Control session; WU15, WU40 and WU60—Warm-up with 15%, 40% and 60% of maximal inspiratory 
pressure (MIP), respectively.
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Inspiratory measurements and inspiratory muscle warm‑up. 
The analysis was conducted by a 
trained researcher who demonstrated the correct performance of the respiratory maneuver. The participants 
remained seated on a chair, wearing a nose clip and a plastic mouthpiece connected to an analogical manovacu-
ometer (± 300  cmH2O; GER-AR, São Paulo, SP, Brazil) used to measure maximal pressures. A small hole (2 mm) 
was introduced in the rigid mouthpiece in order to prevent glottic closure. The participants were instructed to 
complete three to five acceptable and reproducible maximum maneuvers (i.e., differences of 10% or less between 
values), with 1 min interval between  maneuvers43,44. Each inspiratory effort was sustained for at least 1 s, and the 
MIP was considered the highest value between these  attempts45.
After 1 h, a dynamic proposal to characterize the strength of the participants’ IM, the global strength index 
(S-Index), was assessed by an inspiratory threshold (POWERbreathe K5, IMT Technologies Ltd., Birmingham, 
UK), with the participants in standing position and using a nose clip. Thirty dynamic inspirations resistance-
free were performed slowly, with verbal encouragement to inhale a greater air  capacity32. During the protocol, 
breathing pattern curves were monitored by graphic records provided by Breathe-link® software. At the end of 
the test, the algorithm provided the mean and peak values of the S-Index (in units of  cmH2O).
The inspiratory muscle warm-up (IMW) protocol loads were also applied using the inspiratory device POW-
ERbreathe K5. The participants initiated every breath from residual volume and were encouraged to continue 
the respiratory effort until further excursion of the thorax was not possible, with a diaphragmatic breathing pat-
tern. Subsequently, they were instructed to keep the same inspiratory pressure and the breathing pattern curves 
were also monitored by Breathe-link® software. The total protocol was comprised of two sets of 15 inspirations 
with a 1-min rest interval between them. The loads were equivalent to 15% (WU15), considered placebo by 
the  literature32,35,39, 40% (WU40) and 60% (WU60) of MIP. All experimental trials were randomly distributed.
The 30 s all‑out run test and mechanical measurements. 
The 30 s all-out run was performed on a 
non-motorized treadmill (NMT) (Inbramed Super ATL, Inbrasport, Porto Alegre, Brazil), as detailed by Man-
chado-Gobatto et al.10. Two minutes after the IMW or CON protocols, the participants were asked to run at 
maximum intensity for 30 s, tethered by their waist to an inextensible steel cable attached to a load cell (CSL/
ZL-250, MK Controle e Instrumentação Ltda, Brazil) for horizontal force measurement. Other four load cells 
(CSAL/ZL-500, MK Controle e Instrumentação Ltda, Brazil) were positioned under the NMT platform to meas-
ure the vertical force (signal frequency at 1000 Hz). A hall-effect sensor in the frontal axis of the NMT provided 
pulses for velocity acquisition. Therefore, both vertical and horizontal force components were measured during 
the running exercise along with velocity to calculate the power running. The signals were synchronized and the 
product between force and velocity resulted in the running power, with the peak, mean and minimum values 
relativized to body mass. Fatigue index (FI) was also calculated by the following equation: FI = (peak power—
minimum power)/peak power * 100). The system was calibrated on the test days.
Physiological responses. 
Blood lactate concentration and heart rate. For lactate concentrations ([Lac]) 
at rest, post-effort and every 2 min up to 18 min of passive recovery, blood samples (25 µl) were collected from 
the participants’ earlobe with heparinized capillaries, deposited in microtubes (Eppendorf, 1.5 ml containing 
50 µl of 1% sodium fluoride—NaF) and frozen at − 20 °C. The [Lac] were determined by a lactate analyzer (YSI-
2300-STAT-Plus™, Yellow Springs, USA). Throughout the protocols, the heart rate (HR) was constantly recorded 
(at 1 Hz) (Polar V800, Finland). For all variables, the peak, mean and minimum values were calculated (during 
the test we used the 30 s responses, while during passive recovery we considered the mean of 18 min).
Muscle oxygenation by NIRS measurements. 
Total hemoglobin ([tHb] = oxyhemoglobin 
 ([O2Hb]) + deoxyhemoglobin [HHb]) and the equilibrium between oxygen supply and consumption were calcu-
lated using the tissue saturation index (TSI =  [O2Hb]/([O2Hb] + [HHb]) × 100%)46 throughout the experimental 
protocol by two PortaMon devices (Artinis, Medical Systems BV, Zetten, Netherlands) working on the modified 
Beer–Lambert law. Each device has three light source transmitters (with two wavelengths of 760 and 850 nm), 
positioned at 30, 35 and 40 mm from the receiver. The devices were safely fixed and covered to eliminate back-
ground light after shaving and cleaning the skin surface. While one was positioned in the medial part (belly) of 
the  BB10,37,47 of the right arm, considered less active during running, the other was allocated in the VL of the right 
leg (considered more active), 15 cm above the proximal edge of the patella and 5 cm to the external  side10,48–50. 
Skinfolds for BB (3.3 ± 0.2 mm) and VL (11.2 ± 1.2 mm) were less than half the distance between the source and 
the deepest detector (i.e., 20 mm). Different path lengths (DPF) were used for BB (3.78) and VL (3.83)10. The 
signals were smoothed using a  10th order low-pass zero-phase Butterworth filter (cutoff frequency of 0.1 Hz)50, 
and recorded and analyzed on Oxysoft® software (Artinis Medical System, Netherlands). The (Δ)[tHb] in micro-
molar units (μM) was obtained by subtracting these values from the final 30 s of the baseline period of 3 min. 
Examples of NIRS signals in BB and VL muscles of one participant during the four sessions (CON, WU15, 
WU40 and WU60) are displayed in Supplementary file 1, whereas the descriptive graphics of  [O2Hb] and [HHb] 
during and after the 30 s all-out run are in Supplementary file 2.
Statistical analysis. 
All analyses were performed using Statistica software (version 7.0), and the results 
are expressed as mean ± standard error of the mean (SEM). Data normality and homogeneity distribution 
were tested by Shapiro–Wilk and Levene’s test, respectively. Two-way repeated measures analysis of variance 
(ANOVA) was applied to study the effect of IMW (CON x WU15 x WU40 x WU60) and time during the 30 s 
of exercise (on mechanical and physiological responses) as well as the 18 min (every 2 min) of passive recovery 
(on physiological responses) compared to the baseline condition (BL). Three-way repeated measures ANOVA 
investigated the effect of IMW, time, and limb muscles (BB vs VL) on muscle oxygenation during the 30 s all-out 
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run and recovery phase. One-way repeated measures ANOVA was applied to investigate the effects of IMW pro-
tocols on peak, mean, minimum values for force (N/kg), velocity (m/s), running power (W/kg), FI, as well HR 
and [Lac] values. Two-way repeated measures ANOVA (effects of IMW and limb muscles) analyzed the peak, 
mean and minimum values of muscle oxygenation responses. Lastly, post hoc Newman−Keuls test was used to 
detect differences (P ≤ 0.05). The partial eta-squared (ηp
2: 0.01 = small, 0.06 = moderate, 0.14 = large) statistics 
provided estimates of the effect sizes.
Results
Analyses during the 30 s all‑out run. 
The mechanical and heart rate results during the 30 s all-out 
run are shown in Fig. 2. Panel A shows similar running power responses between interventions during the 
30 s all-out run test. The two-way repeated measures ANOVA demonstrated a significant effect of time on this 
parameter  (F(29,1740) = 255.50, P ˂ 0.001, ηp
2 = 0.790). There was an increase in running power in the first second 
until a peak power was reached at approximately 6 s and a consequent decrease after this time for all interven-
tions, without any effects of IMW on the studied parameter. Panels B, C, D and E display the peak, mean and 
minimum values for power, force, velocity and FI, respectively. For these measurements, the one-way repeated 
measures ANOVA revealed an effect of IMW  (F(3,45) = 2.98, P = 0.041, ηp
2 = 0.166) on peak power (Panel B), with 
higher values in WU60 (34.1 ± 1.0 W/kg, P = 0.029) compared to CON (31.2 ± 1.4 W/kg). For mean power (Panel 
B;  F(3,45) = 4.5, P = 0.007, ηp
2 = 0.232), minimum power (Panel B;  F(3,45) = 3.3, P = 0.028, ηp
2 = 0.181), peak force 
(Panel C;  F(3,45) = 3.9, P = 0.013, ηp
2 = 0.208), minimum force (Panel C;  F(3,45) = 5.5, P = 0.002, ηp
2 = 0.269), all IMW 
loads led to higher values in comparison with CON. Additionally, regarding specific IMW effects on mean force 
(Panel C;  F(3,45) = 3.2, P = 0.028, ηp
2 = 0.180), higher values were observed in WU40 (5,6 ± 0.1 N/kg, P = 0.035) and 
WU60 (5.6 ± 0.1 N/kg, P = 0.020) than in CON (5.2 ± 0.1 N/kg), whereas a higher mean velocity value (Panel D; 
 F(3,45) = 3.5, P = 0.022, ηp
2 = 0.190) was obtained for WU60 (4.5 ± 0.1 m/s) than for CON (4.3 ± 0.1 m/s). Panel 
Figure 2.  Mechanical and physiological results observed during the 30 s all-out run under control conditions 
(CON, black color) and after IMW loads at 15% (WU15, blue color), 40% (WU40, green color) and 60% of MIP 
(WU60, red color). ✝ Indicates difference in relation to the control session. (P < 0.05).
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F represents the HR responses second by second during the 30 s all-out run. The two-way repeated measures 
ANOVA pointed to a significant effect of time  (F(29,1740) = 350.76, P ˂ 0.001, ηp
2 = 0.854), yet no effects among 
interventions were detected. The HR constantly increased, reaching the highest values in the last second of the 
test. Furthermore, the peak, mean and minimum HR values were not influenced by IMW (Panel G).
The changes in muscle oxygenation are shown in Fig. 3. Panels A and B illustrate the TSI responses in BB 
and VL, respectively. The three-way repeated measures ANOVA revealed effects of limb muscle  (F(1,120) = 85.83, 
P ˂ 0.001, ηp
2 = 0.419), time  (F(29,3480) = 928.57, P ˂ 0.001, ηp
2 = 0.886) and significant interaction between time 
x limb muscle  (F(29,3480) = 36.39, P ˂ 0.001, ηp
2 = 0.233), but no IMW effects. The peak, mean and minimum 
TSI data (Panel C) were analyzed by two-way ANOVA, which indicated the effect of limb muscle (TSI; peak: 
 F(1,30) = 20.67, P ˂ 0.001, ηp
2 = 0.408; mean:  F(1,30) = 33.49, P ˂ 0.001, ηp
2 = 0.527 and minimum:  F(1,30) = 71.53, P ˂ 
0.001, ηp
2 = 0.705) and IMW only for TSI mean values  (F(1,90) = 3.72, P = 0.014, ηp
2 = 0.110). Despite these findings, 
no interaction between IMW and limb muscles was observed.
The Δ [tHb] for BB and VL are presented in Panels D and E. The three-way repeated measures ANOVA 
showed the effects of limb muscle  (F(1,119) = 15.03, P ˂ 0.001, ηp
2 = 0.112), time  (F(29,3451) = 82.66, P ˂ 0.001, 
ηp
2 = 0.410), and an interaction between time x limb muscle  (F(29,3451) = 16.86, P ˂ 0.001, ηp
2 = 0.124). However, 
no difference was observed through post-hoc analyses (IMW x time x muscle limb). Whereas for peak values 
no differences were observed, for mean  (F(1,30) = 10.00, P = 0.003, ηp
2 = 0.250) and minimum values (Panel F; 
 F(1,30) = 23.73, P ˂ 0.001, ηp
2 = 0.442) a limb effect was detected without interaction with IMW.
Analyses in passive recovery. 
Physiological responses obtained at baseline (BL) immediately after the 
30 s all-out run (T0) and during passive recovery (every 2 min: T2–T18) are displayed in Fig. 4. Panel A shows 
that the curves of blood lactate were similar in all interventions, with a clear effect of time  (F(10,600) = 1053.76, P 
˂ 0.001, ηp
2 = 0.946). A [Lac] peak was observed at 8 min for all participants ([Lac] peak in CON: 17.14 ± 0.62; 
WU15: 16.00 ± 0.51; WU40: 16.11 ± 0.56 and WU60: 16.60 ± 0.65, mM). No effects of IMW were observed. 
[Lac] values did not return to their initial values (BL) after 18 min of recovery, independently of the IMW 
load applied. A similar HR behavior was observed for all conditions (Panel C), with a significant effect of time 
 (F(10,600) = 1646.51, P ˂ 0.001, ηp
2 = 0.965). The HR peak was obtained immediately after the 30 s all-out run (post-
effort, T0), reaching higher values than T2–T18 and decreasing over time. After 18 min, the HR values did not 
Figure 3.  Results of tissue saturation indexes (TSI, Panels A–C) and total hemoglobin ([tHb], Panels D–F) 
in biceps brachii (BB) and vastus lateralis (VL) at each second during the 30 s all-out run on a non-motorized 
treadmill under control conditions (CON, black color) and after the IMW loads with 15% (WU15, blue color), 
40% (WU40, green color) and 60% of MIP (WU60, red color), in line graphs. In bar graphs, the dark colors 
represent the BB, while the light colors correspond to the VL. MIP = maximum inspiratory pressure.
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reach the initial values under any condition. Peak, mean and minimum values for [Lac] and HR are shown in 
panels B and D, respectively. IMW was not able to influence these results.
Changes in muscle oxygenation during passive recovery are shown in Fig. 5. Panels A and B display the 
TSI responses in BB and VL, respectively. The three-way repeated measures ANOVA revealed effects of limb 
muscle  (F(1,120) = 93.23, P ˂ 0.001, ηp
2 = 0.437), time  (F(10,1200) = 643.46, P ˂ 0.001, ηp
2 = 0.843) and a significant 
interaction between time x limb muscle  (F(10,1200) = 32.43, P ˂ 0.001, ηp
2 = 0.213), but no interaction with IMW. 
In all interventions, BB presented lower saturation in relation to BL at T0 and T2, and after 4 min of recovery 
(T4) the saturation returned to BL values. An interesting finding was observed in WU40, which reached higher 
oxygenation values from T4 to T10 compared to BL. In VL, only WU60 at T2 presented different values from 
BL. Moreover, higher saturation was detected in VL than in BB at T0. The two-way repeated measures ANOVA 
revealed an effect of limb muscle on BL  (F(1,30) = 61.16, P ˂ 0.001, ηp
2 = 0.671), peak  (F(1,30) = 17.87, P ˂ 0.001, 
ηp
2 = 0.373), mean  (F(1,30) = 30.06, P ˂ 0.001, ηp
2 = 0.501) and minimum  (F(1,30) = 55.17, P ˂ 0.001, ηp
2 = 0.648) 
values (Panel C) without IWM interaction.
Regarding [tHb] (Panel D‒E), there were a significant effect of time  (F(10,1200) = 44.73, P ˂ 0.001, ηp
2 = 0.272) 
and interaction between time x limb muscle  (F(10,1200) = 2.95, P ˂ 0.001, ηp
2 = 0.024). Compared to BL values, the 
passive recovery time showed lower values for BB at T0 only in WU40, while higher values were noted at T2–T12 
in WU60, subsequently returning to BL values. In VL, differences from BL values were observed at specific 
moments in CON (T2‒T8), WU40 (T18) and WU60 (T4‒T18). IMW strategies did not affect peak, mean and 
minimum [tHb] values in passive recovery (Panel F).
Discussion
To the best of our knowledge, this is the first study dedicated to investigating the effects of different IMW loads 
(15, 40 and 60% of MIP) on mechanical and physiological responses, including oxygenation in more and less 
active muscles, during and after high-intensity, short-duration running exercise. Additionally, we studied these 
acute inspiratory strategies using a shorter protocol (i.e., lower number of exercise repetitions). Our main findings 
revealed some effects of IMW, performed with 2 sets of 15 repetitions with a 1-min rest interval between the sets, 
on the high-intensity running effort and recovery, independently of the load applied. Regarding the mechanical 
parameters, all IMW promoted a significant increase in mean and minimum running power, as well as in peak 
and minimum running force compared to CON. Additionally, when applying specific IMW loads higher values 
were observed for peak power, mean velocity (WU60) and mean force (WU40 and WU60) in relation to CON. 
The physiological responses, including HR and oxygenation in more and less active muscles during the running 
exercise, were not modified by IMW, at least not during the 30 s high-intensity running nor for HR and [Lac] 
in the post-effort phase. By comparing the responses in BB and VL, no differences were observed in mechanical 
and muscle oxygenation during the 30 s all-out test. In passive recovery, higher TSI values for VL were detected 
in the post-effort phase (T0) for all protocols. An interesting finding was observed in WU40, which reached 
Figure 4.  Physiological results of passive recovery immediately after the 30 s all-out run and every 2 min up to 
18 min (mean ± EPM). Results under control conditions (CON, black color) and after IMW protocols with loads 
at 15% (WU15, blue color), 40% (WU40, green color) and 60% of MIP (WU60, red color). # corresponds to 
difference in relation to baseline (BL) for each protocol. Dashed line means no differences among protocols.
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higher oxygenation values from T4 to T10 compared to BL. It can be then suggested that the use of different loads 
of IMW promotes an improvement in performance corroborated by the increased peak, mean and minimum 
mechanical values, but not in the performance assessed second by second. Also, WU40 may improve recovery 
phase with higher oxygenation in BB.
Inspiratory muscle warm‑up and performance. 
Our choice to investigate the effect of different IMW 
loads on the performance of high-intensity, short-duration running exercise was based on previous studies that 
indicate the positive effect of IMW, but used different IWM protocols in intermittent  running27, in Wingate 
 tests33,34, in 100 m freestyle  swimming29, in specific hockey  drills31 and in a simulate judo  match32. Additionally, 
considering the large use of tethered efforts in physical and sports programs together with the significance of 
high-intensity exercise in this context, we focused on the evaluation of the IMW impact on the force, velocity 
and running power of 30 s all-out run sessions using different inspiratory loads. As shown in Fig. 2, the same 
characteristics were observed for the curve of running power throughout the tests, with no differences among 
the IMW load interventions during the 30 s all-out run sessions. As previously mentioned, running power, force 
and velocity were improved by the IMW loads, more specifically the WU40 and WU60, which significantly 
influenced the mechanical variables compared to CON, suggesting an improvement in running performance for 
active participants. Regarding the exercise performance of athletes, the IMW combined with specific warm-up 
was capable of reducing the time in 100 m freestyle  swimming29, treadmill sprint  performance51 and interactions 
among the technical-tactical, physical, physiological, and psychophysiological parameters in a simulated judo 
 match32. Studies that used IMW as the only means of prior effort to main motor task also observed a reduction in 
the sensation of dyspnea and an improvement in the distance walked in one badminton-footwork  test36, as well 
as in one shuttle run  test52. Similarly, Özdal and  colleagues34 observed an increase in peak and relative power in 
Figure 5.  Results of tissue saturation indexes (TSI, Panels A–C) and total hemoglobin ([tHb], Panels D–F) in 
passive recovery immediately after the 30 s all-out run (T0) and every 2 min up to 18 min (T2–T18) compared 
to BL values. Timeline panels show the biceps brachii (BB) and vastus lateralis (VL) under control conditions 
(CON, black color) and after the IMW loads with 15% (WU15, blue color), 40% (WU40, green color) and 
60% of MIP (WU60, red color). Bar graphs present the peak, mean and minimum values (mean ± SEM), with 
dark colors for BB and light colors for VL. MIP = maximum inspiratory pressure. # corresponds to difference 
compared to baseline (BL); * means significantly different from BB. Dashed line means no differences among 
protocols. Colored line means differences in a specific protocol.
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anaerobic Wingate test performed by hockey athletes after IMW (2 × 30 at 40% of MIP with a 2-min rest interval 
between the sets).
Some additional positive effects of IMW on respiratory response and performance in a shuttle run  test27, 
on respiratory muscle  strength28,53, on performance in a knee flexion–extension protocol accomplished in an 
isokinetic test by healthy sedentary  participants41 and on long-distance  test30 were previously observed. On the 
other hand, other studies did not find significant effects of IMW for both active individuals and athletes perform-
ing different types of exercise and  tests39,43,46,53–56. It is important to consider the diversity of the IMW protocols 
when applied to different populations, sports modalities and exercise tests, which makes it difficult to compare 
the results obtained with other findings. Moreover, most investigations do not describe the time interval between 
the IMW sets and the time between the IMW application and the test or main exercise. Knowing that the effects 
of warm-up can be affected by several factors, such as protocol, load, performance level, type of exercise, time 
interval between the conditioning stimulus and the performance testing, etc.57,58, more attention could be paid 
to these aspects. In this sense, we focused on a shorter IMW protocol (2 sets of 15 repetitions with a 1-min rest 
interval between them, concluded 2 min before the running test), using different inspiratory loads in each session 
(without and with 15, 40 and 60% of MIP) applied to active participants. Regarding the respiratory parameters, 
even though our participants did not have any experience with respiratory training or inspiratory warm-up, 
they reached good MIP values (145.6 ± 9.9  cmH2O), similar to Japanese athletes in triathlon and wrestling (light 
category) modalities (145.8 and 147.3  cmH2O, respectively)59.
Inspiratory muscle warm‑up and physiological responses. 
The ventilatory responses in high-inten-
sity exercises can affect the perfusion dynamics of the locomotor muscles and tissue saturation indices, rep-
resenting a limitation of exercise  performance19,20,60. According to the literature, inspiratory muscle warm-up 
can be a strategy to potentialize oxygenation redistribution to more active muscles during physical  exercise35. 
However, improvements in post-effort recovery process remain unexplored. Thus, we measured for the first 
time the physiological responses in exercise and recovery, including oxygenation analysis in more or less active 
muscles (which are relevant to providing oxygen and removing metabolites, respectively). Few scientific inves-
tigations, especially in sports, have been conducted to study the IMW potential to minimize respiratory fatigue 
and improve the oxygenation redistribution in high-intensity  exercise35,40,46,51,61. When studying the oxygena-
tion in the gastrocnemius muscle of female soccer players by submitting them to submaximal cycling test and 
intermittent cycling test, Cheng et al.35 demonstrated that the IMW protocol can enhance oxygen saturation in 
this tissue. However, the authors did not observe changes in performance, possibly due to the lack of specificity 
in the test for these athletes.
In our study, second-by-second NIRS analyses did not reveal the effects of IMW on muscle responses, regard-
less of the load (Fig. 3, Panels A‒B). Despite the evidence of limb muscle effect on TSI peak, mean and mini-
mum values (Fig. 3, Panel C) and [tHb] mean and minimum values (Fig. 3, Panel F), no interaction with IMW 
protocols was observed, indicating a similar behavior in BB and VL oxygenation. Whether the IMW promotes a 
positive effect on the oxygenation  redistribution35, this was not observed in more and less active muscles. Con-
sidering the increased respiratory muscle work and the competition with locomotor muscles for  O2  supply17–20, 
the analysis of inspiratory muscle oxygenation could provide some insight into the oxygenation between these 
muscles. However, the analysis of oxygenation occurs only in accessory and secondary inspiratory  muscles62, 
and it does not directly reflect the oxygenation of the muscle with the greatest potential for oxygen uptake and 
the most affected one by IMW, the diaphragm. Recently, studies addressing the IMW applied to speed skaters on 
ice time trial also did not report any improvement in muscle oxygenation variables in the right VL, with some 
limitations pointed out by the authors, such as leg compression garments and small sample  size40,46. On the other 
hand, in high-intensity  sprint10 and high-intensity  cycling37 a difference in more and less active muscles was 
observed, suggesting adjustments in oxygenation during effort in a tissue-dependent manner.
In order to support the high demand of the respiratory muscles during exercise, the  VO2 and oxygenation 
in this region are increased, and may compromise cardiac output by 14–16% in well-trained  individuals63, thus 
affecting oxygenation distribution to locomotor  muscles64. In high-intensity effort, these locomotor muscles also 
use predominantly anaerobic pathways, resulting in lactate production. According to previous studies, inspiratory 
muscles may play an important role as lactate  consumers21,22. In this context, Lin et al.36 indicated a reduction 
in [Lac] in badminton players after IMW. Regarding [Lac], the peak values observed here (~ 16 mM) confirmed 
their significant anaerobic contribution in the 30 s all-out run. These findings corroborate previous studies on 
exercises characterized by anaerobic  contribution9,10,65,66. In our study, both HR and [Lac] were not affected by 
IMW interventions, and 18 min post-effort in passive recovery was not sufficient to make these physiological 
responses return to baseline values (Fig. 4, Panels A–D).
With respect to passive recovery, a higher decrement in oxygenation was observed immediately after the 
exercise, the so-called post-effort phase (T0), with quick adjustments after T2 for both muscles (Fig. 5). Osawa 
et al.37 reported that tissue oxygenation did not begin immediately after high-intensity cycling effort and that 
deoxygenation occurred for a few seconds. In the present study, TSI percentages started to rise immediately after 
the exercise, and after 4 min (T4) they returned to baseline values (Fig. 5, Panels A‒B). Interestingly, only WU40 
presented higher TSI values in BB from T4 to T10 in relation to BL. We did not perform correlation analyses, 
however, Manchado-Gobatto et al.10 observed a significant correlation between the [Lac] peak and TSI and [tHb] 
values in BB. In this regard, higher TSI in BB suggests an important role of this parameter during recovery since 
 O2 is considered to be essential in oxidative pathways for lactate  clearance67. In VL, only WU60 at T2 reached 
lower values than BL. Despite our initial hypothesis, which proposed that 40% of MIP would redirect oxygena-
tion to VL with a consequent positive impact on mechanical responses during a tethered 30 s all-out run, in 
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addition to a better blood lactate removal during recovery in relation to other loads, we observed that different 
inspiratory loads can improve mechanical parameters and recovery oxygenation.
Recent studies investigated the effects of IMW as a warm-up strategy combined with core warm-ups on recov-
ery period between intermittent exercise and repeated sprints on  NMT51 and on recovery periods of sprints on a 
cycle  ergometer68. Although the authors evaluated recovery, muscle oxygenation was only observed during exer-
cise. In our study, the comparison between BB and VL revealed that only immediately after the 30 s all-out run 
the VL presented higher values for all interventions (Fig. 5, Panels A–B). These results corroborate those reported 
by Manchado-Gobatto et al.10, who did not use inspiratory strategy to improve the running performance. Finally, 
the responses during exercise and recovery are a complex process. Thus, to improve the interpretation of IMW on 
running and recovery, integrative analyses could reveal responses beyond conventional statistics. For example, 
our group recently observed the improvement in the technical and tactical parameters in a judo simulated fight 
using the same shorter IMW protocol used herein, based on a complex network  analysis32. In such study, the 
centrality metrics revealed that the IWM at 15% of MIP favored the interactions among the psychophysiological, 
physical and physiological parameters, while the IWM at 40% of MIP was able to improve performance in the 
judo match. Therefore, our next investigations will be considering these findings.
Furthermore, a recent study indicated NIRS measurements as a future physiological marker, showing no 
significant differences regarding the respiratory compensation  point69. These findings highlight the relationship 
between systemic (i.e., ventilatory) and peripheral (i.e., oxygenation of locomotor and non-locomotor muscles) 
physiological breakpoints. In this sense, future studies should consider respiratory strategies associated with 
the NIRS technique to improve knowledge about the intensity of the training zone. Moreover, the IMW can 
attenuate muscle deoxygenation during  exercise35 and the NIRS technique can contribute to the monitoring of 
oxygenation in clinical  practice70, especially in patients with exercise-induced ischemic pain caused by reduced 
blood flow to the lower  extremities71.
Limitations and strengths
Despite the use of technologies with high-frequency signal acquisition, some limitations regarding our results 
must be addressed. First, the all-out run performed on a NMT has shown reliable results in the scientific 
 literature5,7–10,66,72. However, we did not test the reproducibility of the four IMW interventions – although it is 
safe to say that they exhibited similar results in the 30 s all-out run tests (Fig. 2) with no differences in power 
running among the IMW loads second by second. We chose to investigate a classic anaerobic test (30 s all-out 
run), considering the aerobic component around ~ 18–20% in these  efforts7,13. We observed an effect of IMW on 
the mechanical parameters, which did not result in greater muscle oxygenation differentiation. It is possible that 
by applying another slightly longer exercise protocol or repeated sprints the impact of IMW can be observed on 
both mechanical parameters (second by second) and physiological responses. Additionally, we did not use the 
gas analyzer to investigate the oxygen uptake due to our experimental design, nor investigated oxygenation of 
the inspiratory muscles. Still, we are aware that the association of NIRS measurements and  VO2 exchange would 
improve our data interpretation, but we have not tested whether this equipment can interfere with breathing 
pattern or breath frequency altering the isolated effects of IMW. Another limitation refers to our participants’ 
characteristics, as only healthy active males, non-athletes performed the test. In future studies, we suggest the 
inclusion of female participants, the comparison of IMW with a shorter protocol and the analysis of effects in 
both high-performance athletes and non-athletes in different types of exercises, such as repeated-sprint effort.
The strengths of this study include: (i) the investigation of the inspiratory muscle strategy through a shorter 
protocol applied with methodological rigor, performed with practical and high-quality inspiratory devices, (ii) 
the running effort performed on a non-motorized treadmill able to identify, with high signal capture, minimal 
changes in mechanical variables during high-intensity exercise; (iii) monitoring oxygenation responses in dif-
ferent muscle groups, associated or not with respiratory strategies, allows a more integrative interpretation of 
this variable during effort and also during recovery.
Conclusion
In summary, different IMW loads with a shorter protocol (2 sets of 15 repetitions with a 1-min rest interval 
between sets and 2 min before exercise) applied on high-intensity running exercise suggested an improvement 
in performance corroborated by increased peak, mean and minimum mechanical values, but not in power and 
oxygenation assessed second by second. With respect to muscle oxygenation, these measurements demonstrated 
that the mechanisms by which IMW could possibly exert an effect on performance were not affected by these 
protocols, as all interventions showed similar and rapid adjustments of oxygenation responses during exercise 
demands. Interestingly, during passive recovery WU40 presented a pronounced TSI value for BB, indicating a 
greater availability of  O2 for lactate clearance in a tissue-dependent manner.
Data availability
The data that support the findings of this study are available from the corresponding author on reasonable 
request. Correspondence and requests for materials should be addressed to F.B.M.G.
Received: 7 February 2022; Accepted: 9 June 2022
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Author contributions
F.B.M.G., C.A.G and M.A.M. conceived the main conceptual ideas, designed the project, performed data analysis 
and interpretation, and were responsible for funding acquisition. A.B.M. and F.M.R. conducted data collection, 
analysis and interpretation, and designed figures and table. A.B.M., C.A.G and F.B.M.G. wrote the main text 
of the manuscript. M.A.M. and C.S.H. provided support on inspiratory muscle protocols and performed data 
analysis. All authors reviewed the manuscript and have approved the submitted version.
Funding
São Paulo Research Foundation—FAPESP (2009/08535-5, 2012/06355-2, 2016/50250-1, 2018/05821-6, 
2019/10666-2 and 2019/20894-2), the National Council for Scientific and Technological Development—CNPq 
13
Vol.:(0123456789)
Scientific Reports |        (2022) 12:11223  | 
https://doi.org/10.1038/s41598-022-14616-w
www.nature.com/scientificreports/
(307718/2018-2, 308117/2018-2) and the Coordination for the Improvement of Higher Education Personnel—
CAPES (Finance Code 001). We also thank all the participants for the voluntary participation.
Competing interests 
The authors declare no competing interests.
Additional information
Supplementary Information The online version contains supplementary material available at https:// doi. org/ 
10. 1038/ s41598- 022- 14616-w.
Correspondence and requests for materials should be addressed to A.B.M.
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© The Author(s) 2022

Effects of different inspiratory muscle warm-up loads on mechanical, physiological and muscle oxygenation responses during high-intensity running and recovery.
Marostegan, Anita B,Gobatto, Claudio A,Rasteiro, Felipe M,Hartz, Charlini S,Moreno, Marlene A,Manchado-Gobatto, Fúlvia B