*2.3. Statistical Analysis*

Data are presented as mean ± SD. The normality of data was confirmed using the Shapiro–Wilk test. A two-way repeated-measures ANOVA (group × intensity) was used to compare changes in the involvement of individual breathing sectors and respiratory rate in the intervention and control groups. Significant interactions were examined using Bonferroni adjusted simple main effect post hoc comparisons. An alpha-level of 0.05 was used to assess statistical significance for all comparisons. Subsequently, effect size was determined using Cohen s d. The Pearson correlation coefficient was used to examine relationships between changes in tidal volume and pressure values on the dynamometer. The alpha-level was set to 0.05. The data processing was done in Excel 2016 (Microsoft, Oregon, WA, USA) and Statistica 12 (StatSoft, Tulsa, OK, USA).

#### **3. Results**

Participants carried out the yoga-based breathing program for an average of 13.3 ± 2.8 min per day during the two-month period. In the experimental group, there was a significant increase in the involvement of the abdominal segment during deep breathing and at 2 and 3 W·kg−<sup>1</sup> (*<sup>p</sup>* < 0.05; see Figure <sup>2</sup> and Table 1). The only significant change in thoracic involvement was seen at 3 W·kg−<sup>1</sup> (*<sup>p</sup>* < 0.01). In subclavian respiration, there was no significant change in involvement at any of the intensities, even at rest or at rest during deep resting breathing. In the control group, there was no significant change in the involvement of individual breathing sectors at rest or at any load level (*p* > 0.05; Table 1).

**Table 1.** Average values measured by probes and standard deviations of pressure on individual breathing sectors at rest and at different load intensities in the experimental group (EG) and control group (CG).


Note: \* *p* < 0.05, \*\* *p* < 0.01, Cohen s d: <sup>s</sup> small effect size, <sup>m</sup> medium effect size, <sup>l</sup> large effect size.

> As a result of the breathing exercise intervention, the experimental group experienced a significant reduction (*<sup>p</sup>* < 0.05) of respiratory rate under load 3 and 4 W·kg−1, with medium (4 W·kg−1) or small (rest, deep rest, 2 and 3 W·kg−1) effect sizes. We noted a significant increase of tidal volume at 2 W·kg−1, there are changes with small effect size, during all intensities of load. Minute ventilation and oxygen consumption were not significantly altered (see Table 2). The overall effect of breathing exercise intervention in all phases on changes of respiratory rate and tidal volume was confirmed at level *p* < 0.01.

**Table 2.** Percent change in respiratory rate (RR), tidal volume (VT), minute ventilatory volume (VE) and oxygen consumption (VO2) after breathing exercises intervention versus exercise prior to breathing exercises intervention at different intensities in the experimental group (EG) and control group (CG).


Note: ANOVA: \* *p* < 0.05, \*\* *p* < 0.01, Cohen s d: <sup>s</sup> small effect size, <sup>m</sup> medium effect size.

**Figure 2.** Engagement of breathing sectors at rest (**A**), during deep breathing (**B**), under load 2 W·kg−<sup>1</sup> (**C**), under load 3 W·kg−<sup>1</sup> (**D**), under load 4 W·kg−<sup>1</sup> (**E**) pre and post intervention.

Changes in tidal volume were significantly related to abdominal probe pressure at all intensities (see Table 3).

**Table 3.** Pearson correlation coefficient of change in tidal volume and abdominal probe pressure.


In all intensities, greater abdominal and less subclavian percentage contribution was noted (see Figure 2).

#### **4. Discussion**

The primary finding of the present study was an alternation in breathing patterns at rest, and during cycling exercise at various intensities, in young healthy individuals following an eight-week breathing intervention. This finding corroborates previous research by our group in showing greater and more efficient abdominal contribution to respiration following a breathing intervention [37]. Moreover, it is worthy to note that the respiratory musculature involvement following the intervention was close to what may be recommended [4].

Physical exertion often increases the perception of respiratory effort in healthy people and leads to a feeling of dyspnea. Sports activities, be it intensive, short duration (≥85% of the maximum oxygen uptake) or less intense, longer-lasting duration ("ultramarathon" etc.) can lead to fatigue of the inspiratory and/or expiratory muscles [24]. Moreover, tired respiratory muscles impair athletic performance. During physical activity and sport, work of the respiratory muscles is compounded by greater demand for postural stabilization and movement efficiency [40]. Body stability is impaired when the respiratory muscles are tired, which can increase the risk of tripping or falling [24].

Respiratory therapy is an integral part of treatment for many patients with various diseases. Respiratory contributions have been shown to limit exercise in patients with heart failure. The manner in which the respiratory system limits exercise is due to abnormalities in ventilation, perfusion, or both ventilation and perfusion inspiratory muscle weakness may induce several impairments in both healthy and athletic individuals [22]. Similarly, studies have demonstrated that inspiratory muscle strength also has an important role in the pathophysiology of exercise limitation in several clinical conditions. Indeed, IMT is becoming an effective complementary treatment with positive effects on muscle strength and exercise capacity. More recently, studies have found that maximal inspiratory pressure (MIP) is strongly correlated with VO2 peak in patients after acute myocardial infarction and heart failure, reinforcing the influence of the inspiratory muscles on functional capacity [41]. The exercises primarily reduced end-expiratory lung volume rather than end-inspiratory lung volume, which is constrained by the presence of a thoracic load. Consequently, the training stimuli may be targeting and strengthening the inspiratory muscles throughout an operational range, which may not be utilised during exercise with load carriage. Importantly, previous work has identified that fatigue of the expiratory muscles is not an influencing factor in determining operational lung volumes, despite reduced end-expiratory time and increased peak gastric and esophageal pressures, and it may be more appropriate to assess influences that inhibit flow [42]. In general, the IMT performed at an intensity of 30% MIP resulted in decreased cardiac sympathetic modulation (LF) and increased parasympathetic (HF) at rest in patients with hypertension, heart failure, and diabetes mellitus [43]. Nevertheless, this measure has been questioned as interventions can elicit either complex non-linear reciprocal or parallel changes in either division of ANS, and these complex interactions can influence the calculation and interpretation of LF/HF [44]. However, applying IMT to different diseases, associated with a variety of training protocols, as well as few studies found in the literature, makes the effects of IMT on cardiovascular autonomic control inconclusive. Inspiratory muscle training promotes

changes in cardiovascular autonomic responses in humans [43]. Though inspiratory muscle training seems to improve maximal inspiratory pressure, it remains unclear whether these benefits translate to weaning success and a shorter duration of mechanical ventilation [25].

It is important to note that all participants were encouraged to breathe spontaneously during the testing period to ensure that any observed changes were in fact attributable to the intervention. Interestingly, the observed significant increase in abdominal contribution to breathing was noted at rest and during light/moderate intensities (2 and 3 W·kg<sup>−</sup>1), but not at the greatest load (i.e., 4 W·kg−1). Greater involvement of the thoracic musculature was also observed at lower (i.e., 3 W·kg<sup>−</sup>1) but not the greatest workload. This may be ascribed to greater anaerobic energy contribution at the greatest workload and thus the need for excess ventilation to remove rapidly accumulating CO2 [13,45]. However, of relevance was the observation of a trend towards a reduction in subclavian involvement at the higher workload in the experimental group. At the same time, there was a decrease in respiratory rate, an increase in tidal volume while maintaining the minute ventilation volume and oxygen consumption. Reduced subclavian involvement together with decreased respiratory rate and increased tidal volume at the same minute ventilation volume and same VO2 denotes greater respiratory efficiency and thus greater oxygen availability for mechanical musculature. At the same time, a decrease in respiratory rate also signals a decrease in respiratory work as one of the possible effects of a targeted breathing exercise program. A link has been shown between an increase of pressure on the abdominal probes and an increase of tidal volume. Furthermore, the increased contribution of the abdominal sector to respiration, together with the decreased respiratory rate, and increased tidal volume with the similar minute ventilation, indicates an improved breathing economy [38,46]. This is important as respiratory muscle efficiency is one of the conditions for good performance in endurance.

At rest, and during deep breathing, greater recruitment of abdominal muscles helps to optimize respiratory efficiency and delay the onset of respiratory muscle fatigue. However, during submaximal exercise, a significant alteration in respiratory musculature characterized by a reduction in abdominal and increased subclavian contribution is observed. Our results suggest that while it is possible to manipulate spontaneous breathing patterns during exercise, these benefits may be limited to lighter loads that are likely below the ventilatory threshold. However, reduced respiratory rate following breath training at both low and high workloads may be of benefit across a range of exercise performance disciplines.

The present findings should be interpreted in the context of the population; a greater training effect may be anticipated in adolescents in whom respiratory patterns during exercise are not as well engrained [47]. Like previous research in the field, we strategically selected an eight-week training intervention [48–51]. Future studies to determine the possible benefits of shorter breath training interventions, as well as the persistence of these adaptations if breath training is stopped, are warranted. Other limitations of the present work include our relatively modest sample size as well as that much of the training was performed at-home without direct supervision. Verification of these findings in non-athletic populations and potentially less healthy individuals, such as those with breathing illnesses, would also be of interest.

#### **5. Conclusions**

These data highlight an increased reliance on more efficient abdominal and thoracic musculature, and less recruitment of subclavian musculature, in young endurance athletes following a two-month breathing intervention. More efficient ventilatory muscular recruit at both lower and higher intensities during exercise may benefit endurance performance by reducing oxygen demand of the ventilator musculature and thus increasing oxygen availability for mechanical work.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/jcm10163514/s1.

**Author Contributions:** Conceptualization, P.B., R.M., and D.M.; methodology, P.B., V.B., R.M., D.M., G.J.G., and J.S.; software, P.B. and D.M.; validation, P.B., V.B., R.M., and D.M.; formal analysis, P.B., D.M., and J.S.; investigation, P.B., R.M. and D.M.; resources, P.B., R.M., D.M., and J.S.; data curation, P.B., V.B., and G.J.G.; writing—original draft preparation, P.B., V.B. and G.J.G.; writing—review and editing, P.B., V.B., R.M., D.M., G.J.G., and J.S.; visualization, P.B.; supervision, P.B., V.B., and G.J.G.; project administration, P.B., R.M., D.M., and J.S.; funding acquisition, P.B. and R.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Grant Agency of University of South Bohemia within the framework of Team grant project No. 021/2019/S.

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Ethics Committee of Faculty of Education, University of South Bohemia, Ref. No.: 001/2018, from 19 October 2018.

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study.

**Data Availability Statement:** Data sharing not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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