male 8-week - old wistar rats ( 200    250  g ; specific pathogen - free ( spf ) ; japan shizuoka 
 laboratory center ) were individually housed in cages ( 33    23    23  cm ) under controlled 
 conditions of temperature ( 245    1c ) , humidity ( 55    5  % ) and light ( 12  h light12  h 
 dark cycle ) .
rats were provided with a stock diet ( charles river formula ( crf-1 ) ; oriental 
 yeats co. ltd ) and water ad libitum .
they were accustomed to treadmill 
 running at a 0  % gradient and a speed of 25  m / min for the first 10  min and 85  m / min for 
 the last 10  min ( kn-73 , natsume seisakusho co. ltd ) two times during 1 week of the 
 acclimatisation period .
all procedures were performed in accordance with the animal 
 experimentation guidelines of sugiyama jogakuen university .
milk ( meiji co. ltd ) , sports drink ( pocari sweat ; otsuka co. ltd ) and dextrin ( cyclic 
 cluster dextrin ; esaki glico co. ltd ) were used .
mps was obtained by overnight dialysis of 
 milk in visking membrane tubes ( medical science co. ltd ) .
mes was prepared by mixing kcl , 
 nacl and kh2po4 ( wako pure chemical co. ltd ) .
the composition and 
 osmolality of the experimental solutions are shown in table 1 . in experiment 2 ,
[ 2-c]acetic acid was added to 
 experimental drinks ( cambridge isotope laboratories inc . ) at a concentration of 01  l / ml . 

table 1.components of experimental drinksenergy density ( kj / l)lipid ( g / l)osmolality ( mosm / l)na ( mmol / l)k ( mmol / l)water0019060milk285639255186383sports drink1130034019550mps6310321633mes28560141217383mps , milk protein solution ; mes , milk electrolyte solution .
. a total of eighty - three rats were starved for 20  h from 17.00  hours and were deprived 
 of fluids from 11.00  hours on the day of the experiment .
they ran on a treadmill for 
 60  min ( 11.00 to 12.00  hours ) at a 0  % gradient and a speed of 25  m / min for the first 
 10  min and 85  m / min for the last 50  min .
rats were housed in custom - made metabolism 
 cages for urine collection from 12.00 to 17.00  hours .
average body weight reduction from 
 the baseline values was 31  g and body fluid loss was 12  % of pre - exercise body weight . 

the experimental drinks water , milk , sports drink , mps and mes were orally administered 
 at the dose of 24  % of baseline body weight ( average 60  ml ) using a stomach sonde 
 under diethyl ether anaesthesia at 13.00  hours .
after administration , urine was 
 collected every 1  h for 4  h and was stored below 20c .
urine was collected and weighed , 
 and osmolality and na and k concentrations were measured .
a total of fifteen rats were treated as in experiment 1 except that they were housed in 
 a respiratory metabolic chamber to collect respiratory gas .
the experimental drinks 
 water , milk , mps and sports drink contained [ 2-c]acetic acid at a dose of 
 01  l / ml .
respiratory co2 analysis was performed using an 
 open - circuit metabolic gas analysis system connected directly to a mass spectrometer 
 ( model arco2000 ; arcosystem ) .
room air was pumped through the chambers at a rate of 
 05  litres / min .
expired air was dried in a thin cotton column and then directed to a ms 
 o2/co2 analyser .
the peak time of co2 
 emission was used as the index of gastric emptying rate for each experimental 
 drink 
 .
a total of sixty rats were treated as in experiment 1 and administered the experimental 
 drinks water , milk , mps and sports drink .
blood was collected from the common iliac vein 
 under ether anaesthesia at 30  min , 1  h and 2  h after the administration of drinks . 

osmolality of plasma and urine was measured cryoscopically using an osmometer ( osmomat 
 030 ; gonotec ) .
the concentrations of na and k in plasma and urine 
 were analysed using hplc 
 . briefly ,
na , k , mg and ca ions were separated in a mobile phase buffer 
 ( 075  mm - sulfuric acid , 025  mm - copper sulfate ( ii ) ; ph 24 ) on a 
 super ic - cation column ( 46  mm inner diameter    150  mm ; tosoh co. ) , and ions were detected 
 using an indirect uv method ( 230  nm ) .
plasma aldosterone levels were measured using an 
 elisa kit ( enzo biochem inc . ) .
the between - group comparisons 
 of single - point measurements were made using one - way anova followed by tukey 's 
 post hoc test .
the time course studies of urine osmolality and 
 electrolyte excretion were compared using two - way anova followed by bonferroni 
 post hoc tests .
significance was set at 
 p  <  005 . all statistical analyses were performed using prism 
 software ( windows version 4.03 ; graphpad software ) .
the mean body mass loss during the dehydration protocol was 12  % of the initial body 
 weight .
the 
 volumes of urine excreted each hour over the course of the experiment ( 4 h ) are presented 
 in fig .
drinks ingested following exercise 
 significantly altered cumulative volumes of urine excreted over the course of the 4-h 
 recovery period ( p  <  00001 ) .
urine excretion was significantly 
 less in the milk group compared with that in the water group 
 ( p  <  005 ) .
the volume of urine excreted by rats in sports drink , 
 mps and mes groups was more than that by rats in the water group and less than that by 
 rats in the milk group .
additionally , the volume of urine collected by the mes group 
 drastically increased 3  h after rehydration , although there were no significant changes in 
 urine volume between mes- and milk - administered groups for the initial 2  h of rehydration . 

1.cumulative urine excretion over time after consumption of the five experimental 
 drinks in expt 1 : water ( ---- ) ; milk ( ---- ) ; sports drink ( ---- ) ; milk protein 
 solution ( mps ; ---- ) ; milk electrolyte solution ( mes ; ---- ) .
the right 
 y - axis indicates body fluid retention expressed as a percentage 
 relative to the water group .
values are means , with standard errors represented by 
 vertical bars ( n 13 , 14 , 18 , 19 and 19 for water , milk , sports 
 drink , mps and mes , respectively ) .   mean values with unlike letters 
 were significantly different ( p<005 ) . cumulative urine excretion over time after consumption of the five experimental 
 drinks in expt 1 : water ( ---- ) ; milk ( ---- ) ; sports drink ( ---- ) ; milk protein 
 solution ( mps ; ---- ) ; milk electrolyte solution ( mes ; ---- ) .
the right 
 y - axis indicates body fluid retention expressed as a percentage 
 relative to the water group .
values are means , with standard errors represented by 
 vertical bars ( n 13 , 14 , 18 , 19 and 19 for water , milk , sports 
 drink , mps and mes , respectively ) .   mean values with unlike letters 
 were significantly different ( p<005 ) .
the cumulative volume of urine collected from milk , mps and mes groups was 55 , 31 , and 
 23  % less than that from the water group 4  h after rehydration , respectively .
these 
 percentages reflect the retention of body fluid during the 4-h recovery period ( fig .
furthermore , 
 the sum of body fluid retentions in the mps ( 31  % ) and mes ( 23  % ) groups equalled that in 
 the milk group ( 55  % ) .
thus , milk protein could contribute to 56  % ( i.e. 31/55  % ) of the 
 body fluid retention of milk .
in preliminary studies comparing whole milk to fat - free milk 
 ( data not shown ) and mes ( 2866  kj / l ) to mes without dextrin ( 0  kj / l ; data not shown ) , the 
 energetic content of the drinks showed no significant effect on the volume of urine 
 excreted . there was a significant difference in the urinary electrolyte concentrations after 
 consumption of rehydration solution ( table 2 ) . 

rats in milk and mes groups , whose drinks contain more electrolyte than the other 
 experimental drinks , showed significantly larger urinary na and k excretion compared with 
 that in the water and mps groups at 2  h after rehydration 
 ( p  <  005 ) .
however , urinary na excretion tended to be lower in 
 rats administered milk compared with those administered mes 1  h after rehydration , whereas 
 urinary k excretion was not significantly different between these groups .
table 2.urinary electrolyte excretion and osmolality following rehydration ( expt 1)*(mean values with their standard errors)time after rehydration ( h)1234anova ( p)mean 
 sem 
 mean 
 sem 
 mean 

sem 
 mean 
 sem 
 drinktimedrink    timena ( mmol / l)water20051504390615034000010000105968milk7512204672052929035sports drink450669161132820131mps815058211183419744mes17241203351732222834k ( mmol / l)water20437131502908831430000010000100001milk335686831077566244263sports drink29452379673916524836mps28630284305058541568mes43635591734113326030osmolality ( mosm / kg)water26445088737623028388059793040150952709671milk621914338750158191728846610961sports drink31144923543864429410993571593mps406182842001014539185672421237mes59565927258121737634232679379mps , milk protein solution ; mes , milk electrolyte solution .
mean values with unlike superscript letters were significantly 
 different ( p  <  005).*n 13 , 14 , 18 , 19 and 19 for water , milk , sports drink , mps and 
 mes , respectively .
urinary electrolyte excretion and osmolality following rehydration ( expt 1 ) * ( mean values with their standard errors ) mps , milk protein solution ; mes , milk electrolyte solution .  
mean values with unlike superscript letters were significantly 
 different ( p  <  005 ) .
n 13 , 14 , 18 , 19 and 19 for water , milk , sports drink , mps and 
 mes , respectively .
urine osmolality differed significantly depending on the drink administered 
 ( p  <  005 ; fig .
urine osmolality was significantly higher than that in the water group at 2 
 and 3  h after administration ( p  <  005 ) .
in the mes group , urine 
 osmolality was also significantly higher than that in the water group at 2  h 
 ( p  <  005 ) , but the difference disappeared at 3  h after 
 rehydration . at 3  h of rehydration ,
urine osmolality in mps - administered rats was less 
 than that of the milk group and more than that of the water group .
2.plasma aldosterone levels after consumption of the four experimental drinks in expt 
 3 : water (  ) ; milk ( ) ; sports drink ( ) ; 
 milk protein solution ( ) .
values are means , with standard errors 
 represented by vertical bars ( n 15 ) .  
mean values 
 with unlike letters were significantly different 
 ( p  <  005 ) .
plasma aldosterone levels after consumption of the four experimental drinks in expt 
 3 : water (  ) ; milk ( ) ; sports drink ( ) ; 
 milk protein solution ( ) .
values are means , with standard errors 
 represented by vertical bars ( n 15 )
.   mean values 
 with unlike letters were significantly different 
 ( p  <  005 ) .
gastric emptying of milk was slightly slower than that for other experimental drinks , 
 although this difference was ns .
there were also no differences between gastric emptying 
 times of mps , water and the sports drink ( table 
 3 ) .
table 3.gastric emptying rate of experimental drinks ( expt 2)*(mean values with their standard errors)gastric emptying rate ( min)mean 
 sem 
 water545milk621sports drink507mps529mps , milk protein solution.*n 4 , 3 , 4 , and 4 for water , milk , sports drink and mps , 
 respectively. no significant differences were observed .
gastric emptying rate of experimental drinks ( expt 2 ) * ( mean values with their standard errors ) mps , milk protein solution .
n 4 , 3 , 4 , and 4 for water , milk , sports drink and mps , 
 respectively .  no significant differences were observed . in comparing the two electrolyte - free drinks ( mps and water ) ,
plasma aldosterone levels 
 were significantly higher in the mps group than in the water group . despite the na content 
 of milk , plasma aldosterone levels in rats administered milk were higher than that those 
 in rats administered water .
conversely , plasma aldosterone levels were slightly lower 
 after rehydration with the sports drink than with water because na lowers plasma 
 aldosterone , although this difference was ns ( fig . 
 2 ) .
plasma na concentration levels showed a significant decrease after administration of all 
 the drinks .
however , plasma na level increased again after administration of milk or mps , 
 but not after the sports drink or water ( fig . 

plasma k levels increased immediately after administration of all the 
 drinks , but decreased to basal levels 3  h after the sports drink or water .
in contrast , 
 plasma k concentrations remained high in both the milk and mps groups ( fig .
3.plasma sodium ( a ) and potassium ( b ) levels after consumption of the four 
 experimental drinks in expt 3 : water ( ---- ) ; milk ( ---- ) ; sports drink ( ---- ) ; 
 milk protein solution ( -- ) .
values are means , with standard errors represented by 
 vertical bars ( n 15 )
.   mean values with unlike 
 letters were significantly different ( p  <  005 ) .
plasma sodium ( a ) and potassium ( b ) levels after consumption of the four 
 experimental drinks in expt 3 : water ( ---- ) ; milk ( ---- ) ; sports drink ( ---- ) ; 
 milk protein solution ( -- ) .
values are means , with standard errors represented by 
 vertical bars ( n 15 )
.   mean values with unlike 
 letters were significantly different ( p  <  005 ) . from 30  min to 2  h after administration , plasma osmolality in rats administered water and 
 the sports drink decreased significantly compared with that in rats administered milk and 
 mps ( p  <  005 ) . at 4  h after administration ,
plasma osmolality in 
 the mps group was maintained at the initial level , while a decrease in plasma osmolality 
 was observed in the other three groups , but was much slower in the milk group than that in 
 the sports drink or water groups ( fig .
4.plasma osmolality after consumption of the four experimental drinks in expt 3 : 
 water ( -- ) ; milk ( ---- ) ; sports drink ( ---- ) ; milk protein solution ( -- ) .
values 
 are means , with standard errors represented by vertical bars ( n 5 ) . 
 
mean values with unlike letters were significantly different 
 ( p  <  005 ) .
plasma osmolality after consumption of the four experimental drinks in expt 3 : 
 water ( -- ) ; milk ( ---- ) ; sports drink ( ---- ) ; milk protein solution ( -- ) .
values 
 are means , with standard errors represented by vertical bars ( n 5 )
. 
  mean values with unlike letters were significantly different 
 ( p  <  005 ) .
rehydration after exercise is associated with smooth water absorption and total body fluid 
 retention . in several previous studies ,
total body fluid retention has been shown to be 
 superior in milk compared with that in isotonic electrolyte drinks 

 . while milk is considered an effective post - exercise rehydration drink , 
 the effect of milk protein itself on total body fluid retention has not been estimated . 
 here
, we showed that the cumulative volume of urine excreted following rehydration in rats 
 with milk , mps and mes was 55 , 31 and 23  % less , respectively , than that with water 4  h 
 after rehydration ( fig .
1 ) . the 32  % difference 
 ( 5523  % ) between milk and mes may reflect the effects of milk protein on total body fluid 
 retention , as both of these experimental drinks have equal electrolyte content and energy 
 density .
this difference of 32  % was nearly equal to the 31  % relative retention of urine in 
 the mps group . given that mps contains milk proteins but not electrolytes , milk protein 
 itself appears to contribute 56  % ( 31/55  % ) of the body fluid retention effect of milk .
interestingly , urine volume from mes - administered rats rapidly increased 3  h after 
 rehydration , while it was not different from that from the milk group for the first 2  h of 
 rehydration ( fig .
this discrepancy between mes 
 and milk may be related to differences in the composition of these two experimental drinks , 
 with the most typical candidate substance being milk protein .
. 
  also showed differences in urine volume after 3  h of rehydration with 
 energy- and electolyte - matched carbohydrate and carbohydrate  milk protein solutions . 

together , these findings suggest that milk protein is more effective in fluid retention than 
 carbohydrate , although carbohydrate also exerts a mild influence on body fluid 
 retention 
 .
although previous studies have indicated the effectiveness of milk protein on body fluid 
 retention , the mechanisms behind this have not been determined 

 .
these reports indicate that protein clotting in the stomach delays the 
 gastric emptying of milk .
the half - emptying time of milk was estimated to be 14  % slower 
 than that of the sports drink 
 .
we showed that the gastric emptying rate of mps is not different from 
 that of water or the sports drink , while the gastric emptying rate of milk is 14  % slower as 
 described by a previous report earlier ( table 3 ) . 
 to estimate the gastric emptying rate , we used c - labelled acetic acid as a 
 marker in a respiratory gas analysis system .
absorption and oxidation of acetic acid are 
 rapid , and can be utilised as a marker of gastric emptying rate 
 .
the gas analysis system used in this study was optimised for the small 
 volume of respiratory gas emitted by rats , and was sufficiently accurate for the purpose of 
 the study 

 .
the results show that milk protein itself does not lower the gastric 
 emptying rate , and also indicates that this is slowed by interactions between protein and 
 electrolyte fractions . in a previous human study , urine osmolality 2  h after milk intake was about three to nine 
 times higher than that following intake of water and a sports drink 

 . the same applied to the observation in the present study .
urine 
 osmolality after administration of milk was maintained at a higher level than that after 
 administration of the sports drink or water ( table 
 2 ) .
given that the elevation of urine osmolality was greater than that of na , k or 
 cl , another plasma component derived from milk may contribute to this elevated osmolality . 

urinary na excretion in the mes group was higher than that in the milk group during the 
 first 1  h of rehydration .
on the other hand , differences in urinary k excretion between rats 
 administered mes and milk were not observed during the first hour of rehydration , but were 
 significantly different at 3 and 4  h of rehydration ( table 2 ) .
we have demonstrated that raised plasma aldosterone contributes to the total body fluid 
 retention 30  min and 1  h after administration of milk ( fig . 
 2 ) .
however , as na ingestion suppresses plasma aldosterone levels 
 , the difference between the milk and water groups was ns .
the 
 renin  angiotensin  aldosterone system is , from an evolutionary viewpoint , a well - preserved 
 regulatory system with many morphological and physiological similarities in all vertebrate 
 groups from fish to mammals 
 .
elevation of plasma aldosterone level causes the reduction of renal na 
 excretion and the increasing of renal na retention 
 .
aldosterone secretion is known to be regulated by various factors such as 
 the renin 
2 ) in the mps group compared with 
 the water group 30  min after administration .
although the mechanism that induces aldosterone 
 secretion is unclear , a 5-d high - protein diet ( 2  g / kg body weight / d ) caused increased plasma 
 renin , aldosterone and vasopressin levels in human subjects 
 .
similar changes in vasoactive hormones may have occurred in the present 
 study , as animals were orally administered approximately 1  g / kg body weight at a time after 
 exercise .
elevation of plasma aldosterone positively affects body fluid retention by conserving 
 plasma na 
 .
we showed that plasma aldosterone and na levels in the mps group were 
 higher than in rats administered water or the sports drink 13  h after administration of the 
 drink ( fig .
in contrast to the mps group , plasma na levels in the water group decreased linearly for 3  h 
 after administration . at this time point
plasma na levels were highest in the milk group , 
 followed by the mps group , the sports drink group and the water group .
, plasma osmolality 
 in the mps group was maintained at a higher level than that in the water and sports drink 
 groups for 2  h after rehydration ( fig .
4 ) . as mps 
 contained no electrolytes , this elevated plasma osmolality and na retention can not be 
 explained by the electrolyte content of the drink .
therefore , higher plasma osmolality in 
 the mps group can be partially explained by the higher na retention ( fig .
3(a ) ) and by other plasma components derived from milk , such as 
 amino acids or their metabolites .
most previous studies on body fluid retention have been conducted in human subjects by 
 measuring the changes in body weight 

 .
although animal studies have several limitations in predicting the 
 effect of milk protein solution on body fluid retention for human subjects , in this study , 
 total body fluid retention was assessed using small laboratory animals .
of interest is 
 whether there is a specific component in milk that affects body fluid retention . given that 
 it is difficult to obtain a large amount of effective fractions , experimental models using 
 small laboratory animals are useful in evaluating the effects of small amounts of effective 
 fractions on fluid retention . as precise urine collection is difficult in the small 
 laboratory animal , we developed a protocol to measure fluid retention in rats , which 
 requires precise measurement of urine volumes to avoid the confounding effect of body weight 
 on volumes of faeces and urine . in conventional urine collection cages ( metabolism cage )
, 
 the separation of urine and faeces achieves a urine recovery rate of 64  % . to improve this
the authors consider that the effects of anaesthesia on the experimental results were 
 negligible .
inhalational anaesthetics influence renal function by reducing cardiac output 
 and systemic blood pressure , and affecting the concentration of aldosterone , angiotensin ii , 
 atrial natriuretic peptide and vasopressin levels 
 . by means of anaesthesia by ether for 30 
min , urine volume was reported 
 to decrease by half in a human study 
 . on the other hand , in the present study , as anaesthesia was performed 
 for rapid oral administration , the aspiration of ether was within 2  min .
therefore the 
 influence of ether anaesthesia on the observation in the present study was negligible . in summary ,
milk protein was responsible for 56  % of the body fluid retention after milk 
 consumption .
milk protein caused elevated plasma aldosterone levels and suppressed the 
 reduction of plasma osmolality without delaying gastric emptying time .
these data show that 
 milk proteins maintain plasma osmolality and suppress body fluid loss .
 milk is an effective post - exercise rehydration drink that maintains the net positive 
 fluid balance . 
 however , it is unclear which components are responsible for this effect . 
 we 
 assessed the effect of milk protein solution ( mps ) obtained by dialysis on body fluid 
 retention . 
 milk , mps , milk electrolyte solution ( mes ) , sports drink and water were 
 administered to male wistar rats at a dose of 6  ml / rat after treadmill exercise . 
 total 
 body fluid retention was assessed by urine volume 4  h after administration of hydrating 
 liquids . 
 the rate of gastric emptying was evaluated by a tracer method using 
 13c - labelled acetate . 
 plasma osmolality , na and k levels , and urinary na and k 
 were measured by hplc and osmometry , respectively . 
 the gastric emptying rate was not 
 delayed by mps . during 4  h of rehydration , 
 cumulative urine volumes differed significantly 
 between treatment groups ( p  <  005 ) with 49 , 22 and 34  ml from 
 water- , milk- and mps - fed rats , respectively . 
 thus , mps elicited 50  % of the total body 
 fluid retention of milk . 
 plasma aldosterone levels were significantly higher in mps- and 
 milk - fed rats compared with water - fed rats . 
 plasma osmolality was maintained at higher 
 levels in mps - fed rats than in water- and mes - fed rats ( p  <  005 ) . 
 
 cumulative urine na excretion was also suppressed in the milk- and mps - fed groups compared 
 with the mes - fed group . 
 our results demonstrate that mps obtained by dialysis clearly 
 affects net body water balance without affecting gastric emptying after exercise . 
 this 
 effect was attributed to retention of na and water , and maintenance of plasma 
 osmolality . 
Experimental methods
Results
Discussion