with the rapid increase in the proportion of elderly in japan , the proportion of adult 
 diseases such as cancer , ischemic heart disease , cerebrovascular disease , and diabetes 
 mellitus has also increased , now accounting for about 60% of the causes of mortality1 , 2 . 

exercise plays a crucial role in the prevention of adult disease and is recommended to 
 maintain or improve health3 , 4 .
it has been reported that a certain intensity of exercise is required for improvements in 
 health and physical strength5,6,7 .
at least a moderate 
 exercise intensity is required to improve the health of adults6 , but comorbidities in the elderly may restrict their ability to 
 exercise . therefore , exercise should be prescribed at a suitable intensity for the elderly , 
 i.e. , the intensity at which the benefits of exercise can be gained but risks of exercise 
 avoided . the balance between benefit and safety varies with the physical condition of the 
 individual but
is considered to occur at a lower intensity in elderly people with existing 
 disease than in healthy young adults . therefore , exercise intensity in the elderly must be 
 carefully adjusted .
anaerobic threshold ( at ) has been proposed as an objective criterion for precise adjustment 
 of exercise intensity8 . at
is the exercise 
 intensity just after which energy production by anaerobic metabolism is added to aerobic 
 metabolic energy production during incremental exercise .
it is estimated by the lactate 
 threshold ( lt ) , which is the workload at which the blood lactate concentration abruptly 
 increases , and by the ventilatory threshold ( vt ) , which is the workload at which the rate of 
 pulmonary minute ventilation abruptly increases nonlinearly during a progressive exercise 
 test9 , 10 .
it has 
 been reported that both thresholds are safe and effective criteria11 , 12 .
however , both 
 thresholds have disadvantages ; specifically , the gas analyzer used to measure expired gas 
 for determination of vt is expensive and is limited to use in a laboratory setting , and 
 blood collection for determination of lt is invasive .
one is 
 the double - product breakpoint ( dpbp)13 , 
 or the point at which double product ( dp ) , the product of heart rate and systolic blood 
 pressure , abruptly increases during a progressive exercise test .
the dpbp can be calculated 
 from the intersection of two regression lines , and its validity has already been established 
 based on the high correlation between dpbp and at ( r = 0.87 , p < 0.001)14,15,16 .
another method for estimating at is heart 
 rate threshold ( hrt ) , which uses only heart rate17 , 
 18 .
hrt is obtained from the 
 relationship between running speed and heart rate . the running speed 
heart rate relationship 
 is linear from low to submaximal speeds and curvilinear from submaximal to maximal speeds . 

the speed of transition from the linear to the curvilinear phase coincides with the 
 beginning of a sharp accumulation of blood lactate18 .
however , it has been reported that the dpbp method is inferior to 
 expired gas analysis in detecting at14 
 and that the hrt occurs at a higher workload than at19 .
for this reason , it is necessary to develop a more sensitive 
 indicator or to create a method combining a number of indicators to improve the ability to 
 detect at and thus prescribe suitable exercise to subjects with reduced exercise 
 tolerance . both systolic blood pressure and heart rate , the components of dp , are controlled by the 
 sympathetic nervous system .
the plasma catecholamine concentration , which reflects 
 sympathetic nervous system activity during incremental exercise , has been shown to start to 
 rise abruptly at a workload similar to the lt .
in addition , it has been reported that the 
 plasma catecholamine concentration correlates with the blood lactate concentration20 , 21 .
this suggests that dp may reflect changes in sympathetic nervous 
 system activity and blood lactate concentration through the plasma catecholamine 
 concentration .
therefore , it is possible to detect the at more precisely if an index is used 
 that is a more sensitive reflection of sympathetic nervous system activity .
the proposed 
 index of sympathetic nervous system activity is salivary -amylase , an enzyme in saliva that 
 has been reported to be controlled by the sympathetic - adrenal - medullary system22 , 23 
 and to be a sensitive indicator of the activity of the sympathetic nervous system24 .
therefore , it is possible to detect 
 changes in blood lactate concentration indirectly by measuring salivary -amylase , a 
 measurement that can be performed quickly under test conditions .
investigated 
 the relationship between the salivary threshold ( tsa ) , the workload at which salivary 
 -amylase abruptly increases , and the lt by measuring salivary -amylase and blood lactate 
 concentrations during a multistage incremental exercise25 .
the authors concluded that tsa had a high correlation with the lt 
 ( r = 0.95 , p < 0.001 ) , and the detection rate was 80% .
the results suggested that the 
 high correlation between the tsa and lt , as well as the ease of measuring salivary 
 -amylase , makes tsa a valid and useful estimate of at .
moreover , no report has investigated the relationship 
 between tsa and other methods of estimating at
. determination of whether the detection rate 
 is improved by combining tsa and dpbp would provide meaningful information for exercise 
 prescription , particularly for elderly people with preexisting disease .
the purpose of this study was to assess the validity of salivary -amylase as a method for 
 estimating at quickly and to clarify the relationship between salivary -amylase and dpbp in 
 order to establish a way to adjust exercise intensity to a safe and effective range .
the subjects of this study were 11 healthy young adults ( 23.8  1.8  years , height 173  
 5.0  cm , body weight 64.6  6.3  kg , body mass index 21.7  1.3 
none of the 
 subjects reported any neurological or vestibular disorders , orthopedic conditions , or oral 
 cavity diseases before participating in this study .
we used an electrically braked cycle ergometer ( aerobike 75xl , combi co. ltd . ,
the timing of the experiment ( 6:00 p.m. to 8:00 p.m. ) was 
 chosen to minimize the influence of circadian variation in salivary -amylase26 .
since 
 the activity of salivary -amylase can be influenced by alcohol , medications , food , and 
 caffeine , all subjects were instructed not to drink any alcohol the day before measurement 
 and not to consume food or caffeine 2 hours before measurement . after a rest period of 3  min on the ergometer
, subjects were instructed to try to maintain 
 a cadence of 50  rpm while the workload level was increased by 20 watts per 3  min beginning 
 with 10 watts .
the exercise test was continued until subjects could no longer maintain the 
 prescribed cadence due to fatigue or until completion of 2 stages following that at which at 
 was observed . oxygen consumption ( vo2 ) , carbon dioxide production ( vco2 ) , and 
 ventilatory equivalent ( ve )
were measured using a breath - by - breath gas analyzer ( ae-300 , 
 minato medical science , osaka , japan ) .
systolic blood pressure and heart rate were measured during the last 30 
 seconds of each stage of the incremental exercise test .
heart rate was measured 
 using an electrocardiogram monitor ( bsm-2401 , nihon kohden , tokyo , japan ) .
the dpbp was 
 determined using a computer algorithm as follows : the linear regression lines of dp as a 
 function of workload were calculated for all possible divisions of the data .
dpbp was 
 determined by choosing the intersection , or breakpoint , of the 2 lines representing the 
 minimum residual sum of squares from among various intersections of the 2 lines .
salivary -amylase was measured using a portable salivary amylase analyzer ( salivary 
 amylase monitor , nipro , osaka , japan ) .
it was verified that within the analyzer s linear 
 range ( 10230 ku / l ) , this handheld monitor s accuracy ( r = 0.989 ) , precision 
 ( coefficient of variation < 9% ) , and measurement repeatability ( range 3.1% to + 3.1% ) 
 approached those of a more elaborate laboratory - based automated clinical chemistry analyzer 
 ( olympus america inc . , center valley , pa , usa ) .
salivary -amylase was measured from saliva 
 samples collected during the last 30 seconds of each stage of an incremental exercise test 
 by inserting the reagent test strip directly into the subject s oral cavity .
the mask for 
 expired gas analysis was briefly removed to obtain the saliva sample and then replaced after 
 saliva collection .
workload at the at , tsa , and dpbp were compared using one - way anova to 
 investigate the difference in workload between each index .
pearson s correlation 
 coefficients were used to assess the relationship between workload at the at and tsa and 
 between the at and dpbp .
in addition , with tsa-1 ( or dpbp-1 ) representing 1 workload prior 
 to that corresponding to tsa ( or dpbp ) , tsa-2 representing 2 workloads prior , tsa-3 
 representing 3 workloads prior , and so on , tsa , tsa-1 , tsa-2 , and tsa-3 and workloads for 
 dpbp , dpbp-1 , dpbp-2 , and dpbp-3 were compared by one - way anova with repeated measures to 
 identify the dynamics of salivary -amylase and dpbp associated with an incrementally 
 increasing workload .
subject characteristicsage ( yr)23.8  1.8height ( m)1.73  0.05weight ( kg)64.6  6.3bmi ( kg / m)21.7  1.3hr at rest ( beats / min)70.0  11.4hr at at ( beats / min)116.2  16.4vo2 at at ( ml / min)1,170.2  189.3vo2/w at at ( ml / minkg)18.1  2.3salivary -amylase at rest ( ku / l)22.2  27.5salivary -amylase at tsa ( ku / l)40.0  18.1dp at rest ( mmhg  beats / min)8,740.4  1,994.7dp at dpbp ( mmhg  beats / min)14,868.2  3,153.1data presented as mean  sd .
bmi , body mass index ; at , anaerobic threshold ; dp , 
 double product ; dpbp , double - product breakpoint ; hr , heart rate ; tsa , salivary 
 threshold ; w , watt . in the present study , at could be determined in all subjects ( 100% ) .
however , 
 tsa and dpbp could not be detected in 2 of the subjects ( detection rate of 82% for both 
 methods ) because the increase was not abrupt enough to identify 2 regression lines clearly . 
 in the following analysis , therefore , data for the subjects whose tsa and dpbp could not be 
 detected were excluded .
the results of one - way anova revealed that there was no significant 
 difference among workloads at the at , dpbp , and tsa ( table 2table 2 .
power output at at , tsa , and dpbpat ( w)70.0  28.3tsa ( w)61.1  28.5dpbp ( w)65.6  21.9at , anaerobic threshold ; dpbp , double - product breakpoint ; tsa , salivary threshold ; w , 
 watt ) .
bmi , body mass index ; at , anaerobic threshold ; dp , 
 double product ; dpbp , double - product breakpoint ; hr , heart rate ; tsa , salivary 
 threshold ; w , watt at , anaerobic threshold ; dpbp , double - product breakpoint ; tsa , salivary threshold ; w , 
 watt there were significant correlations between at and tsa ( r = 0.951 , p < 0.01 ) and between 
 at and dpbp ( r = 0.940 , p < 0.01 ) .
the results of one - way anova with repeated measures 
 identified the significant main effects of salivary -amylase ( f1.58 , 12.6 = 
 24.0 , p < 0.01 , partial  = 0.75 ) and dp ( f1.34 , 10.3 = 44.7 , p 
 < 0.01 , partial  = 0.85 ) , and post hoc analysis using bonferroni s test was 
 executed .
the results showed that there were significant differences between tsa and tsa-1 
 ( p < 0.05 ) , between tsa and tsa-2 ( p < 0.01 ) , and between tsa and tsa-3 ( p < 0.01 ) . 

however , there were no significant differences between tsa-1 and tsa-2 , between tsa-1 and 
 tsa-3 , or between tsa-2 and tsa-3 .
the purpose of this study was to assess the validity of tsa as a workload index by 
 clarifying the relationship between at , which was measured by analysis of expired gas , and 
 tsa , which represented the workload at which salivary -amylase abruptly increased .
we also 
 investigated the relationship between dpbp , which has been established as a method for 
 quickly estimating at , and tsa in order to assess the usability of tsa as another method for 
 quickly estimating at .
the results of this study indicate significant correlations between at and tsa ( r = 0.951 , 
 p < 0.01 ) .
furthermore , a significant difference was not observed in the workload at the 
 at and tsa .
this result suggests that tsa has validity as a method for estimating at 
 quickly , which confirms results reported by calvo et al25 .
yamamoto et al . investigated the activity of the parasympathetic 
 and sympathetic nervous systems during exercise using heart rate variability .
the authors 
 revealed that the indicator of parasympathetic nervous system activity decreased 
 progressively from rest to a workload equivalent to 60% vt and that the indicator of 
 sympathetic nervous system activity increased only when exercise intensity exceeded vt28 .
these results suggest that the activity 
 of the sympathetic nervous system changed at the vt , which was equivalent to the at , and 
 that at might be estimated by monitoring the activity of the sympathetic nervous system . 

moreover , it has been reported that the salivary -amylase concentration increases with 
 increased physical activity , such as treadmill exercise29 , running30 , 31 , and cycle exercise24 , 32 .
. 
 investigated salivary -amylase during walking , jogging , and running and reported that 
 jogging and running increased salivary -amylase but that walking did not affect salivary 
 -amylase24 .
this result confirmed that 
 the increase in salivary -amylase was caused by physical activity exceeding a certain 
 workload ( i.e. , the at ) .
chatterton et al . also investigated the correlation between 
 salivary -amylase and plasma catecholamines and showed significant correlations between 
 them ( r = 0.64 for norepinephrine and r = 0.49 for epinephrine)24 .
this result directly indicates that salivary -amylase 
 reflects the activity of sympathetic nervous system .
since salivary -amylase is an index 
 that sensitively reflects the activity of the sympathetic nervous system and the activity of 
 the sympathetic nervous system is caused by workloads above the at , it is suggested that 
 tsa , which is the point of inflection of salivary -amylase , is a good index by which to 
 estimate at .
the results of the present study also indicated that tsa was at least as good as dpbp as a 
 method for estimating at quickly because the correlation between the at and tsa was 0.951 ( p 
 <
this 
 result suggests that salivary -amylase reflects the activity of the sympathetic nervous 
 system more sensitively than dp , the components of which are controlled by the sympathetic 
 nervous system .
spence et al . compared the systolic and diastolic blood pressure responses 
 to exercise before the at with those after the at by using a cycle ergometry33 .
the authors concluded that the rate of 
 increase of systolic blood pressure significantly increased after the at .
tanaka et al . also 
 demonstrated that the slope of a regression line of systolic blood pressure and heart rate 
 increased after the at by comparing the slope of a regression line of systolic blood 
 pressure and heart rate before and after the at13 .
however , other researchers reported that the slope of a regression 
 line of systolic blood pressure and heart rate may not increase after the at .
riley et al . 
 investigated the relationship between dpbp and at , as determined from analysis of expiration 
 gas , by measuring systolic blood pressure and heart rate during an exercise test14 .
their results indicated that , although 
 dpbp and at are strongly correlated , the rates of increase of systolic blood pressure and 
 heart rate do not always increase after the at .
in the study of riley et al . , the rate of 
 increase of systolic blood pressure increased after the at for some subjects but that for 
 heart rate did not ; other subjects indicated the opposite . furthermore , conconi et al . 

researched the relationship between hrt and lt by obtaining the hrt , which was calculated 
 from running speed and heart rate18 .
the 
 results indicated that there was a strong correlation between hrt and lt and that increased 
 running speed involved an increase in heart rate .
however , the rate of increase of heart 
 rate after the hrt decreased , which is in contrast to the results of tanaka13 .
both riley et al.14 and conconi et al.18 demonstrated that changes in systolic blood pressure and heart rate 
 before and after the at differed among individuals .
dp is highly correlated with at because 
 it consists of 2 variables , so that even if only 1 variable shows unique changes , the other 
 variable is able to change in a complementary manner . however , using the product of 
 variables that vary among individuals may lead inaccurate estimation of at .
salivary 
 -amylase is therefore superior to dp in the degree of correlation to at because salivary 
 -amylase can measure the activity of the sympathetic nervous system using only 1 
 variable .
there are other ways in which tsa is superior to dpbp for estimation of at .
to calculate 
 dp , blood pressure and heart rate must be measured , but blood pressure measurement 
 temporarily restricts subjects physical activity . additionally , measurement of heart rate 
 requires equipment such as an electrocardiograph monitor , thus restricting the measurement 
 environment .
in contrast , measurement of salivary -amylase requires only insertion of the 
 reagent test strip directly into the subject s oral cavity to collect saliva samples without 
 the need to interrupt activity , and it can be performed in various environments . thus , 
 salivary -amylase estimates at simply and has the advantages of easier measurement and more 
 sensitive reflection of sympathetic nervous system activity than can be achieved with 
 dp .
interestingly , the results of the present study suggest that a more correct estimation of 
 at can be achieved by combining tsa and dpbp . in this research , although at was identified 
 in all subjects using expired - gas analysis , tsa and dpbp were unable to be determined in 2 
 subjects . however , because data allowing determination of dpbp were always collected , 
 combining tsa and dpbp allowed identification of at in all subjects .
this finding suggests 
 that the variables salivary -amylase , systolic blood pressure , and heart rate are 
 controlled by the sympathetic nervous system but that the expression of these variables in 
 vivo differs among individuals .
therefore , inclusion of all the variables in the estimation 
 of at may allow a more precise estimation . at estimation using salivary -amylase
is useful 
 for subjects in whom tsa can be identified even if this is the only method used because tsa 
 and at are strongly correlated .
however , the combination of dpbp and tsa might be desirable 
 for some subjects , since tsa was unable to be identified in about 18% of our study subjects . 

while it is better to measure salivary -amylase to estimate at because of its superiority 
 to dpbp in the degree of correlation with at , if tsa is not able to be determined , dp can be 
 calculated to complement the detection of at . as a result , at can be determined with a 
 detection rate as high as that for expired - gas analysis .
this result is in line with the 
 study of calvo et al.25 , who mentioned 
 that differences among subjects in their hydration state before reporting to the laboratory 
 or in the contribution of each salivary gland to the production of total saliva explained 
 the inability to detect tsa in 20% of their subjects . in this study , although we encouraged 
 subjects to drink water before measurement and allocated a point under the tongue for saliva 
 collection in order to minimize these influences , the detection rate was not improved . 

further studies are needed to improve the precision of measurement of salivary -amylase 
 itself .
the results of this study clarified that tsa is a useful index for estimating at during an 
 incrementally increasing workload .
tsa was not able to be detected in 2 subjects , but in 
 such cases , the rate of determination of at can be improved by combining tsa with dpbp . 

determination of at using salivary -amylase is superior to other methods because the 
 measurement device is easily transportable , making measurement possible in various 
 environments , and because of the noninvasiveness of the technique .
we believe that the 
 ability to adjust exercise to the optimal intensity in various environments using salivary 
 -amylase can make exercise safer and more effective .
   [ purpose ] the purpose of this study was to clarify the validity of salivary -amylase as 
 a method of quickly estimating anaerobic threshold and to establish the relationship 
 between salivary -amylase and double - product breakpoint in order to create a way to 
 adjust exercise intensity to a safe and effective range . 
 [ subjects and methods ] eleven 
 healthy young adults performed an incremental exercise test using a cycle ergometer . 
 during the incremental exercise test 
 , oxygen consumption , carbon dioxide production , and 
 ventilatory equivalent were measured using a breath - by - breath gas analyzer . 
 systolic blood 
 pressure and heart rate were measured to calculate the double product , from which 
 double - product breakpoint was determined . 
 salivary -amylase was measured to calculate the 
 salivary threshold . 
 [ results ] one - way anova revealed no significant differences among 
 workloads at the anaerobic threshold , double - product breakpoint , and salivary threshold . 
 
 significant correlations were found between anaerobic threshold and salivary threshold and 
 between anaerobic threshold and double - product breakpoint . 
 [ conclusion ] as a method for 
 estimating anaerobic threshold , salivary threshold was as good as or better than 
 determination of double - product breakpoint because the correlation between anaerobic 
 threshold and salivary threshold was higher than the correlation between anaerobic 
 threshold and double - product breakpoint . 
 therefore , salivary threshold is a useful index 
 of anaerobic threshold during an incremental workload . 
INTRODUCTION
SUBJECTS AND METHODS
RESULTS
DISCUSSION