muscle fatigue involved in inspiration , such as diaphragm muscle , external intercostal 
 muscle , or parasternal intercostal muscle , induces a shortness of breath resulting in 
 impaired exercise tolerance1 , 2 .
in addition , diminished respiratory muscle strength 
 results in the reduced efficiency of coughing to remove airway secretions ( phlegm ) , which 
 increases the risk of atelectasis or pneumonia after surgery or prolonged bed rest3 , 4 .
age - related physiological changes in the respiratory system , such as decrease in pulmonary 
 elasticity , fusion of the sternal bone and costal cartilage , or increases in thoracic 
 kyphosis , augment the reduction of respiratory muscle strength and respiratory function5 .
indeed , in japan , pneumonia has recently 
 overtaken cerebrovascular disease as the third leading cause of death , reflecting the aging 
 of society .
thus , it is essential to maintain respiratory function , including respiratory 
 muscle strength , for a healthy daily life , particularly for the elderly . as a part of health promotion activities ,
aquatic exercise has generally been accepted in 
 the clinical settings of rehabilitation and various sports facilities6 , 7 .
people with 
 obesity8 , joint diseases9 , or lumbago10 , 11 benefit from aquatic 
 exercise , wherein they can avoid unnecessary exercise load on the joints because of 
 self - weight using buoyancy and can perform exercise more safely . in addition , they can 
 efficiently strengthen the muscles of the upper and lower extremities using water viscosity 
 and pressure in aquatic exercise9,10,11,12 . breathing underwater
requires great effort mainly because of the following two aspects : 
 first , blood volume shifts into the chest cavity because of the increased venous return from 
 the lower extremities ; second , inflexibility of the chest wall and diaphragm shifts toward 
 the cranial side because of the hydrostatic pressure resulting into restricted pulmonary 
 compliance13,14,15 . regarding the effect of 
 water depth on respiratory function , the pulmonary vital capacity ( vc ) , forced expiratory 
 volume during the first second ( fev1.0 ) , and functional residual capacity ( frc ) are 
 decreased during water immersion at the clavicular or cervical level16,17,18 .
furthermore , de andrade et al . reported that the decrease 
 in the maximum inspiratory muscle strength during water immersion was greater when the water 
 level was at the clavicle than at the xiphoid process because of the higher water pressure 
 at the clavicular level19 .
therefore , 
 aquatic exercise , such as swimming or walking in water , is an ideal exercise for health 
 promotion , which can add an aspect of resistance training through water viscosity to aerobic 
 exercise . in addition , the specific circumstance of resistance to chest expansion during the 
 inspiratory phase may be useful for developing respiratory muscle strength .
previous studies 
 have reported that exercise in water for 810 weeks improves the respiratory muscle strength 
 and exercise tolerance of healthy individuals , and patients with chronic obstructive 
 pulmonary disease and spinal cord injury20,21,22 . 

however , it has not been clarified how the water depth during exercise in water affects 
 respiratory muscle strength .
accordingly , the hypothesis was built that inspiratory muscle strength would decrease after 
 aquatic exercise training by muscle fatigue depending on the water depth .
thus , we 
 investigated , 1 ) the effect of water immersion at different water depths on respiratory 
 function , and 2 ) the effect of inspiratory muscle training during water immersion at 
 different water levels on respiratory muscle strength .
sedentary , healthy male college students aged between 20 and 22 were recruited for this 
 study .
subjects who had a history of respiratory or cardiovascular disease , hypertension 
 ( resting systolic blood pressure ( bp )  140 and/or diastolic bp  90 ) , diabetes , obesity 
 ( body mass index ( bmi )  30 ) , or smoking habits were excluded .
the applicants who met the 
 inclusion criteria participated in the study after familiarizing themselves with the 
 experimental protocol , e.g. the spirometry measurement method and inspiratory load breathing 
 ( ilb ) .
this study was approved by the institutional review board of osaka city university 
 graduate school of medicine ( approval no .
2629 ) , and registered with the university hospital 
 medical information network - clinical trial registry
this study 
 also conformed to the principles of the declaration of helsinki , and written informed 
 consent was obtained from all of the subjects prior to the experiment .
the subjects were required to 
 attend the laboratory on three separate days for three trials at different water depths , the 
 umbilical level ( ul ) , the 4th rib level ( rl ) , and the clavicular level ( cl ) trials , in a 
 random order , with at least 3 days separating each trial . in all sessions , a depth - variable 
 water bath ( aquaexmill ocu1 , sanplatec , osaka , japan ) was used , and the water temperature 
 was maintained at 32  1.0 ( sd )  c , the  insensible temperature , to minimize the stress on 
 the body23 , 24 . at the beginning of each session , subjects sat immersed in the water bath with the depth at 
 the umbilical level for 5  min , and then the respiratory function and respiratory muscle 
 strength were evaluated as baseline measurements .
next , subjects sat immersed at one of 3 
 different water levels ( ul , rl , or cl ) . after an additional 5  min , the measurements were 
 repeated for the purpose of evaluating the effects of water immersion at the different 
 depths on the respiratory function and respiratory muscle strength .
then , the subjects 
 performed 15-min ilb in the sitting position , followed by the continuous evaluation of 
 respiratory muscle strength immediately , and 5  min , and 10  min after they had completed ilb 
 immersed in water at the umbilical level in the sitting position .
the water levels were 
 adjusted by changing the height of the chair . the respiratory function and respiratory 
 muscle strength after ilb
the subjects conducted 15  min breathing at a respiratory rate of 15 breaths 
 per  min , synchronized with a metronome , during which the inspiratory muscle load was 
 adjusted to 30% of the maximum inspiratory pressure ( pimax ) at baseline using an inspiratory 
 loading device ( threshold imt , philips , usa ) .
the load of 30% of pimax during ilb was 
 adopted , because it is commonly used as the load in the settings of respiratory muscle 
 training for healthy subjects25 .
the 
 subjects were asked to take normal breaths by maintaining their tidal volumes ( tvs ) as in 
 usual respiration at rest , not deep breaths , during the ilb .
tidal volume ( tv ) , vc , inspiratory capacity ( ic ) , inspiratory reserve volume ( irv ) , and 
 expiratory reserve volume ( erv ) were measured as indices of respiratory function using a 
 spirometer ( as-507 , minato , osaka , japan ) .
pimax and maximum expiratory pressure ( pemax ) in 
 the oral cavity were also evaluated using a sthenometer attached to the spirometer ( aam337 , 
 minato , osaka , japan ) according to the method of black and hyatt26 and used as surrogate indices of inspiratory and 
 expiratory muscle strength , respectively .
chest circumference ( cc ) was measured with a 
 measuring tape at the xiphoid process level .
the rate of change of each parameter of respiratory function and respiratory muscle 
 strength from baseline was calculated using the following equation : rate of 
 change ( % ) = [ ( a measured value)(baseline value ) ] / ( baseline value )  
 100 weight and height were measured before the first experiment . body mass index ( bmi 
 in  kg / m ) was calculated as body weight ( kg ) divided by height ( m ) squared .
all statistical analyses were performed using stat view statistical software package 
 ( ver5.0 , sas , cary , nc , usa ) .
data were expressed as the mean  standard deviation ( sd ) 
 unless otherwise indicated .
one - way analysis of variance ( anova ) with repeated measurements 
 and subsequent multiple pairwise comparisons ( scheffe method ) was performed to evaluate the 
 effects of water immersion at different water depths on the rate of change in the 
 respiratory function and respiratory muscle strength from baseline .
the effects of water 
 depth and ilb on change and the rate of change in the respiratory function and respiratory 
 muscle strength were examined using two - way ( trial  time ) anova with repeated measurements . 
 in the case of a significant time or trial effect , post - hoc multiple pairwise comparisons 
 ( dunnett method ) were performed .
eight applicants , aged 21.3  0.5  years , who met the inclusion criteria were enrolled in 
 the present study .
their mean values of height , weight , and bmi were 1.73  0.1 m , 64.4  
 7.5  kg , and 21.5  1.5  kg / m , respectively .
changes in the parameters of respiratory function and respiratory muscle strength following 
 water immersion in each trial are shown in table 
 1table 1.the effect of water immersion on respiratory function and respiratory muscle 
 strengthulrlclbaselineafter immersionbaselineafter immersionbaselineafter immersionvc ( l)4.10.74.10.64.00.53.90.54.00.53.60.5tv ( l)0.70.20.70.20.70.40.70.40.70.30.80.3ic ( l)2.40.62.40.62.30.52.50.42.50.62.90.8erv ( l)1.60.41.60.41.70.51.40.51.50.40.80.5irv ( l)1.70.61.70.61.70.41.90.31.80.42.10.7cc ( cm)76.85.176.75.176.15.275.95.176.85.275.95.1pemax ( cmh2o)104.422.8106.533.1101.520.8100.226.1105.325.898.724.9pimax ( cmh2o)96.524.392.419.992.127.889.627.597.323.690.124.1values are means  sd.ul : umbilicus ; rl : 4th - rib ; cl : clavicle ; vc : vital capacity ; tv : tidal volume ; ic : 
 inspiratory capacity ; erv : expiratory reserve volume ; irv : inspiratory reserve volume ; 
 cc : chest circumference ; pemax : maximal expiratory pressure ; pimax : maximal 
 inspiratory pressure . there were no significant changes in any parameter due to water immersion in 
 each trial . when we compared the rates of change in each parameter among the three trials , 
 as shown in table 2table 2.the effect of water immersion at different water depths on the rate of change in 
 respiratory function and respiratory muscle strengthrate of change ( % )
ulrlclvc0.81.31.71.29.71.0tv0.12.65.46.58.46.7ic1.70.810.74.114.15.2erv1.21.617.16.146.38.1irv2.22.014.45.617.28.0cc0.10.10.20.71.10.1pemax2.13.61.23.16.32.3pimax4.32.42.73.57.32.4values are means  sem . * p < 0.05 , vs. ul .  p < 0.05 , vs. rl.abbreviations are as table 1 . , the decreases in vc ( ul : 0.8  1.3 , rl : 1.7  1.2 , cl : 9.7  1.0% , p = 0.01 
 for cl vs ul , p = 0.03 for cl vs rl ) , erv ( ul : 1.2  1.6 , rl : 17.1  6.1 , cl : 46.3  8.1% , 
 p = 0.01 for cl vs ul , p = 0.02 for cl vs rl ) , and cc ( ul : 0.1  0.1 , rl : 0.2  0.7 , cl : 
 1.1  0.1% , p = 0.04 for cl vs ul , p = 0.04 for cl vs rl ) from baseline were significantly 
 greater in the cl trial than in the rl and the ul trials .
the increase in ic was also 
 significantly greater in the cl trial than that in the ul trial ( ul : 1.7  0.8 , rl : 10.7  
 4.1 , cl : 14.1  5.2% , p = 0.02 for cl vs ul , p = 0.12 for cl vs rl )
. the rates of changes in 
 the other parameters of respiratory function and muscle strength did not show significant 
 differences among the trials .
ul : umbilicus ; rl : 4th - rib ; cl : clavicle ; vc : vital capacity ; tv : tidal volume ; ic : 
 inspiratory capacity ; erv : expiratory reserve volume ; irv : inspiratory reserve volume ; 
 cc : chest circumference ; pemax : maximal expiratory pressure ; pimax : maximal 
 inspiratory pressure values are means  sem . * p < 0.05 , vs. ul .  p < 0.05 , vs. rl .
changes in respiratory muscle strength following ilb in each trial are shown in table 3table 3.the effect of inspiratory load breathing at different water levels on respiratory 
 muscle strengthbaseline0 min after ilb5 min after ilb10 min after ilbpimax ( cmh2o)ul96.524.389.720.998.623.499.422.8rl92.127.888.425.694.224.5100.222.3cl97.323.684.423.299.921.4100.525.4pemax ( cmh2o)ul104.422.8114.428.3102.723.5109.023.3rl101.520.894.323.1103.124.799.825.7cl105.325.8102.221.1103.820.0105.716.0values are means  sd .
abbreviations are as table 1 .. there were no significant differences in these parameters among three trials . 
 regarding the rate of change in respiratory muscle strength following ilb , compared to the 
 baseline , pimax was reduced immediately after ilb in the cl trial , and the reduction was 
 significantly greater than in the rl and the ul trials ( ul : 7.0  1.5% , rl : 6.7  4.8% , 
 cl : 14.1  4.1% , p = 0.04 for cl vs ul , p = 0.03 for cl vs rl ) . on the other hand ,
pemax was not affected by ilb in any of the trials , and there was no difference 
 in the rate of change in pemax among the three trials .
in the present study , the decreases in vc and erv during water immersion were significantly 
 greater in the cl trial compared to those in the other trials .
reported that 
 vc and forced expiratory volume in 1  s during water immersion reduced in proportion to the 
 depth of the water , and that the reduction became significant when the depth was at the 
 cervical level16 .
dahlback and buono et 
 al . also reported that vc was significantly decreased by water immersion at the clavicle 
 level27 , 28 .
kurabayashi et al . reported that cc was reduced by 0.5  cm with water immersion at cervical 
 level depth , compared to that before submersion29 . in the present study ,
the reduction in cc was significantly 
 greater in the cl trial than in the other trials , and this is also consistent with the 
 results of kurabayashi et al .
therefore , it is likely that the chest wall is compressed by 
 the hydrostatic pressure of water immersion to the clavicular level or deeper .
speculation 
 has emerged that the abdominal wall as well as the chest wall is compressed by hydrostatic 
 pressure during water immersion resulting in cranial shift of the diaphragm , which would 
 reduce the compliance of the chest cavity , or vc .
a cranial shift in the diaphragm and 
 reduced compliance of the chest cavity would also minimize the alveolar size at the 
 end - expiratory phase , which may decrease erv , and , in contrast , increase ic . in the present study ,
neither pimax nor pemax were affected by water immersion , regardless 
 of the depth of the water . in regard to pimax , our results were consistent with the findings 
 of schoenhofer et al.30 who reported that 
 pimax was decreased by water immersion to the clavicular level compared to the baseline 
 before submersion , but that the decrease was not statistically significant .
it is generally 
 recommended that the measurement of pimax is performed at the maximum expiratory phase , 
 based on the theory of the length - tension relationship31 . in short ,
the maximum expiratory phase is determined by the 
 balance between expiratory muscle contraction and the opposing elastic dilatation pressure 
 of the lung and thorax . in this phase ,
the inspiratory muscles and the diaphragm are 
 extended to the utmost and demonstrate maximal tensile strength to generate maximal 
 inspiratory pressure with the support of the elastic dilatation pressure of the lung and 
 thorax .
water immersion could assist the subjects to easily retain maximum expiratory phase , 
 and it may be the reason why pimax was not impaired by water immersion in the present study . 
 regarding pemax , our results support the findings of de andrade et al.19 who reported that pemax during water immersion did not 
 differ regardless of the depth of the immersion .
primarily , submersion accompanied by 
 decreases in vc and cc may be unfavorable conditions for measuring pemax from the 
 perspective of the length - tension relationship cited above .
however , it is possible that 
 hydrostatic pressure against the entire thorax in the cl trial may have assisted expiration 
 resulting in minimal influence of immersion on pemax . to the best of our knowledge ,
no previous report has investigated the influence of water 
 depth during submerged forced respiration on respiratory muscle fatigue . in the present 
 study ,
the decrease in pimax immediately after ilb was significantly greater in the cl trial 
 than that observed in the other trials . in the cl trial , it is possible that hydrostatic 
 pressure against the entire thorax may have added an excessive load on the inspiratory 
 muscle besides the original load of 30% of pimax during ilb .
in addition , it is possible 
 that an extra inspiratory load was elicited by reduced alveolar compliance following 
 increased venous return under the influence of hydrostatic pressure on the lower 
 extremities .
this may be why the greatest decrease in pimax following ilb was found in the 
 cl trial ; it reflected more severe inspiratory muscle fatigue compared to the other 
 trials . on the other hand , although it is considered that even in the rl trial , hydrostatic 
 pressure against the lower part of thorax affected the expansion of the chest cavity , at 
 least in part , there was no significant decrease in pimax following ilb in the rl trial , as 
 well as in the ul trial .
de andrade et al . showed that neither water immersion to the depth 
 of iliac crest nor the xiphoid process level reduced vc compared to that measured on dry 
 land19 .
their findings suggest that 
 immersion to the depth of the xiphoid process level or shallower does not yield enough 
 hydrostatic pressure on the chest wall or venous return to the mediastinal space to impair 
 the expansion of the chest cavity .
therefore , immersion to ul or rl in our study was 
 unlikely to have additionally loaded the inspiratory muscles . in the present study ,
the decrease in pimax immediately after ilb in the cl trial had 
 completely recovered at 5  min after ilb .
our results support the findings of suzuki et al . 
 who reported that pimax was transiently decreased immediately after resistance load 
 respiration training , but the decrease was no longer observed 5  min after the cessation of 
 the training32 .
approximately 60% of the 
 muscle contained in the diaphragm is comprised of red muscle fibers33 , which are characterized by fatigue resistance and 
 endurance strength .
this may be a reason why fatigue of the inspiratory muscle induced by 
 ilb was quickly recovered in our cl trial .
in contrast to pimax , pemax showed no change from 
 baseline following ilb in any trial . in the present study ,
furthermore , hydrostatic pressure against the 
 chest wall could possibly have assisted expiratory muscles to contract .
therefore , our 
 immersed ilb may have resulted in no remarkable fatigue of the expiratory muscles .
the results of this study suggest that the deeper the immersion depth , the greater the 
 fatigue of the inspiratory muscles . generally , training - associated factors which cause 
 muscular fatigue , such as metabolic stress or local hypoxia , are necessary for eliciting 
 muscular hypertrophy and strengthening34 . 

therefore , the results of this study have important implications for exercise in water for 
 patients with chronic respiratory diseases , in which the strengthening of respiratory 
 muscles is widely accepted as a therapeutic strategy .
regardless of the presence of a 
 chronic disease , from a health promotion perspective , our results will be useful for 
 determining the depth of water for aquatic exercise .
however , it is not clear whether 
 training producing greater inspiratory muscle fatigue results in greater muscle strength .
it 
 also has to be determined whether the results of our study of healthy young adults can 
 wholly be applied to patients with chronic respiratory diseases whose respiratory muscles 
 are already fatigued , even at rest .
in addition , the water resistance during aquatic 
 exercise varies according to the kinetic rate , i.e. , a lower kinetic rate correlates with a 
 smaller water resistance in aquatic exercise .
it is possible that respiration in water at 
 low frequency reduces the load against the chest wall in aquatic respiration exercise . 

therefore , it is necessary to develop effective aquatic exercise protocols for strengthening 
 respiratory muscles .
first , the values of pimax and pemax had high 
 variation among individuals . therefore , the small number of subjects may not have been 
 enough to verify the influence of ilb on respiratory muscle fatigue .
furthermore , our 
 subjects performed ilb with a prescribed inspiratory load and respiratory rate .
however , 
 subjects were only asked to maintain their tvs with normal respiration at rest .
although we 
 found no significant difference in tvs during water immersion among the trials , it is not 
 certain whether the subjects could keep tvs during ilb constant across the trials . 

therefore , there is a possibility that differences in tvs influenced the magnitude of 
 inspiratory muscle fatigue . in summary ,
forced respiration during the deepest level of water immersion caused the 
 greatest inspiratory muscle fatigue in healthy young men .
further studies are needed to 
 develop an exercise regimen that can be utilized in the field of sports or clinical settings 
 that makes the most of the characteristics of submersion and aquatic exercise .
 [ purpose ] the purpose of the present study was to evaluate the effect of water immersion 
 at different water depths on respiratory function and the effect of inspiratory load 
 breathing ( ilb ) during water immersion at different water depths on respiratory muscle 
 strength evaluated by maximum inspiratory and expiratory pressures ( pimax and pemax , 
 respectively ) . 
 [ subjects ] eight healthy men participated randomly in three trials . 
 [ methods ] 
 all sessions were conducted with the participants in a sitting position immersed 
 in a water bath . 
 we evaluated respiratory function , pimax and pemax during submersion at 
 three different levels of water depth ( umbilicus ; 4th - rib ; or clavicle , cl ) and after 
 subsequent 15-min ilb . 
 [ results ] decreases in vital capacity and expiratory reserve volume 
 from baseline by water immersion were significantly greater in the cl trial than those in 
 the other trials . in the cl trial , 
 pimax was immediately reduced after ilb compared to 
 that at baseline , and the reduction was significantly greater than those in the other 
 trials . 
 pemax was not affected by ilb in any of the trials . 
 [ conclusion ] forced 
 respiration during deeper water immersion caused greater inspiratory muscle fatigue in 
 healthy young men . 
INTRODUCTION
SUBJECTS AND METHODS
RESULTS
DISCUSSION