continuous blood supply to active muscles is necessary to maintain exercise . in patients 
 with cardiovascular diseases ,
it has been suggested that synchronization between the heart 
 beat and locomotor rhythm [ cardiac - locomotor synchronization ( cls ) ] can optimise blood flow 
 to muscles and minimise cardiac load1 , 2 .
the mechanism of this phenomenon is 
 speculated to be peak intra - arterial pressure due to cardiac contraction occurring at the 
 lowest phase of the intramuscular pressure cycle .
niizeki reported evidence for 
 physiological cls using computer - assisted bilateral thigh - cuff occlusion and suggested that 
 cls may be associated with the improved perfusion of exercising muscles2 .
if cls has the physiological significances described above , 
 cls has the potential to improve the exercise capacity of patients with cardiovascular 
 diseases and thus may be useful in rehabilitation programs .
however , the physiological 
 significance of cls for muscle blood perfusion during walking , running and cycling , which 
 are usually performed in physical therapy , has not yet been clarified in healthy subjects . 

therefore , we examined whether cls has physiological significance for lower leg muscle blood 
 perfusion during walking to obtain evidence of physiological cls in healthy subjects .
eleven healthy men ( mean  sd , height : 167.8  4.5  cm , weight : 56.1  5.0  kg , age : 21.3  
 2.2  years ) were included in this study after obtaining their informed consent .
this study 
 was approved by the ethics committee of seirei christopher university ( approval number : 
 09008 ) .
physical activity and alcohol and caffeinated beverage consumption were prohibited 
 for 24  h before testing .
drinking and eating , except water , were also prohibited for 3  h 
 before testing .
we determined each subject s treadmill load ( treadmill speed and grade ) at which their 
 heart rate ( hr ) was maintained at approximately 120 beats / min ( bpm ) .
the target heart rate 
 was derived from the 5070% of estimated maximal heart rate , which is the formula of optimal 
 heart rate during exercise for heart failure patients in japan .
subjects walked on a 
 treadmill ( autorunner ar-200 , minato medical science co. , ltd . ,
osaka , japan ) , with 
 gradually increasing treadmill speed within the range in which subjects can walk ( limited at 
 6.0  km / h ) .
when the load was insufficient , the treadmill grade was increased until the 
 target hr was achieved .
subjects walked at the determined treadmill speed and grade for at 
 least 5  min to confirm appropriate load .
subjects then rested for at least 15  min during which they were instrumented for data 
 collection .
electrodes for electrocardiography ( ecg ) , a foot switch sensor and a 
 near - infrared spectroscopy ( nirs ) probe were placed on the chest , right heel and posterior 
 surface of the lower left leg , respectively .
thereafter , subjects were instructed to perform 
 two treadmill protocols in a random order . in one , subjects walked at the frequency of their 
 hr to induce cardiac - locomotor synchronization ( cls protocol ) . in the other , subjects walked 
 at their preferred pace ( free protocol ) .
both protocols were performed at the individual s 
 predetermined treadmill load for 20  min .
the first 10  min was the warm - up period , and it was 
 followed by 10  min of measurement . as the treadmill load was fixed for each subject , the 
 effort was identical in both protocols .
subjects rested in a sitting position for at least 
 15  min between protocols . to achieve cls in the cls protocol , subjects walked at their predetermined load for 5  min 
 and then for 5  min with a buzzer signal generated by an ecg monitor ( bedside monitor 
 bsm-2400 series life scope 1 , nihon kohden corp . ,
after their hr reached a steady state , they walked in synchrony 
 with the buzzer signal for 10  min , and data was collected during this period . in the free 
 protocol , subjects walked at their predetermined load for 10  min and then at their preferred 
 pace for 10  min and data were collected during the latter 10-min walking period . thus , 
 identical treadmill loads and exercise times were established for both protocols . the r - r interval ( rri )
the ecg signal was amplified and band - pass filtered between 10300  hz to 
 distinguish r waves of the qrs complex and to suppress movement artifacts .
the ecg signal 
 was digitised at s sampling frequency of 1 khz using a personal computer - based system ( chart 
 5 for windows , ad instruments , shanghai , china ) equipped with an analogue - to - digital 
 converter ( ml880 powerlab 16/30 , ad instruments ) .
the heel contact interval ( hci ) was 
 measured by a foot switch sensor ( inline foot contact sensor , noraxon ) on the right heel . 

the foot switch signal was collected at a sampling frequency of 1.5 khz using a personal 
 computer - based system ( myoresearch xp , noraxon ) . as ecg and foot switch signals
were 
 recorded using separate computers , ecg and hci recordings began at same time .
in addition , a 
 near infrared spectrophotometer ( omega monitor ; bom - l1 trw , omega wave , tokyo , japan ) was 
 used to monitor tissue oxygen saturation .
using these three wavelengths , it is possible to differentiate between oxygenated and 
 deoxygenated haemoglobin .
the sum of oxygenated and deoxygenated haemoglobin reflects the 
 total amount of haemoglobin ( total hb ) , which can be interpreted as blood volume in the 
 underlying tissue3 .
the nirs signal is 
 generally used with artery or venous occlusion to examine tissue blood flow and tissue 
 metabolism4 , 5 .
it was not possible to compress the subjects lower extremities , 
 because the focus of this study was muscle blood perfusion when cls occurred during walking . 

therefore , the index of muscle blood perfusion during walking was defined as total hb only . 

a probe with a dedicated 
 light - occluding holder was attached to the posterior surface of the lower left leg section 
 with the greatest diameter with surgical tape and an elastic bandage .
rri and hci data were translated into text files and opened in microsoft office excel 2007 
 ( redmond , wa , usa ) .
each successive hci signal was halved to estimate the foot switch rate 
 for both legs .
the time at which each r wave occurred and the onset of each step cycle were 
 calculated by adding the first onset time and interval times . to obtain the relative phase 
 relationship between the cardiac and locomotor rhythms ( r , h )
, the time 
 ( tr ) at which r r wave occurred in a one - step cycle was calculated 
 from the onset of the step cycle , where tr is the time of the r marked event . 

the onset of the step cycle was defined as th . the relative phase 
 ( r , h ) of the r heartbeat in a one - step cycle was calculated as 
 follows : where r and h are integers . the synchronization index ,  , was calculated to quantify the strength of phase locking 
 between the heartbeat and step cycle as follows : where n indicates the number of consecutive data samples .
 was calculated from 60-point 
 windows ( approximately 30  s epochs ) with a sliding window of 10 points .
mean  was 
 calculated as the average of the entire measurement period of each protocol .
the fractional 
 points were rounded down when points of relative phases were insufficient at the end of 
 calculations .
 was restricted to 0    1 , and a higher value indicates stronger phase 
 locking between the two rhythms .
a surrogate data technique was also used to determine the probability of entrainment 
 between the cardiac and locomotor rhythms6 .
the surrogate data were generated by randomly shuffling the original 
 order of raw hci data , while successive rris remained the same to create a new phase 
 relationship between the two rhythms .
the phase relationships between the heartbeat and 
 locomotor activity rhythm were destroyed because of the random order of hci .
if two rhythms 
 had an exact phase relationship , a relative phase similar to the original relative phase 
 would occur in the surrogate data .
the average  value of original data was compared with 
 that of surrogate data calculated from an identical observation window .
mean hr , mean step rate ( sr ) and total hb in the lower left leg muscles were calculated 
 over the final 10  min of each protocol to investigate the cls physiological response .
cls occurrence was defined 
 as  exceeding 0.3 , where  is the difference in  between the cls and free protocols 
 ( cls  free ) , because  values show substantial individual 
 variability even during cls induction7 . in 
 subjects
whose  exceeded 0.3 ,  values were compared between the two protocols using the 
 paired t - test to determine whether phase synchronization occurred during the cls protocol . 

in addition , the  values of the original and surrogate data of both protocols were compared 
 using the paired t - test to determine whether phase synchronization occurred by entrainment . 
 for subjects
whose  exceeded 0.3 , mean hr , mean sr and total hb were compared between the 
 two protocols using the paired t - test to examine the effect of cls on muscle blood perfusion 
 during walking .
furthermore , for all subjects , the relationship between  and total hb 
 ( % ) , calculated as the ratio between total hb in the cls and free protocol , using pearson s 
 product - moment correlation coefficients was investigated .
all statistical analyses were 
 performed using spss version 19 for windows software ( spss japan , inc . , tokyo , japan ) .
in 9 of 11 subjects , the difference in the synchronization index ,  , between the cls and 
 free protocol (  ) exceeded 0.3 , indicating the occurrence of cls during the cls protocol in 
 the majority of subjects . in subjects
whose  exceeded 0.3 , the mean  value was also 
 significantly greater in the cls protocol than in the free protocol ( 0.54  0.16 vs. 0.06  
 0.06 , n = 9 , p < 0.05 ) and that of the surrogate data ( 0.54  0.16 vs. 0.24  0.08 , n = 
 9 , p < 0.05 ) , indicating that phase synchronization was the result of heartbeat 
 entrainment by locomotor activity .
in contrast , the mean  value of the free protocol did 
 not differ from that of the surrogate data ( 0.06  0.06 vs. 0.05  0.05 , n = 9 , not 
 significant ) , indicating that phase synchronization did not occur in the free protocol . in subjects
whose  exceeded 0.3 , mean hr and mean sr did not differ significantly between 
 the two protocols ( mean hr : 122  6.5 vs. 120  7.1 beat / min , mean sr : 122  6.3 vs. 124  
 7.9 step / min , n = 9 , not significant ) .
however , despite the identical treadmill load of the 
 two protocols , total hb during the cls protocol was significantly higher than that during 
 the free protocol ( 17.8  2.6 vs. 17.5  2.7 10 pieces / mm , n = 9 , p 
 < 0.05 ) .
for all subjects , correlation analysis revealed a significant relationship 
 between  ( range , 0.160.65 ) and total hb ( range , 98104% ) ( r = 0.64 , n = 11 , p < 
 0.05 ) .
the main finding of the present study was that total hb in the lower leg muscles increased 
 with the  value during walking .
furthermore , the  value of the cls protocol was 
 significantly greater than that of both the surrogate data and the free protocol , indicating 
 that the difference in the physiological data between the two protocols possibly shows a 
 physiological response when cls occurs .
the increased lower leg muscle blood perfusion 
 during walking suggests that this entrainment can enhance exercise efficiency .
total hb increased significantly during the cls protocol compared with that during the free 
 protocol , despite the identical treadmill load .
in addition , a positive correlation was 
 observed between total hb and  , strongly suggesting a causal relationship between the 
 increase in total hb and cls occurrence .
a possible explanation for this is the occurrence 
 of an optimal phase relationship between arterial and intramuscular pressures , leading to 
 improved muscle blood perfusion . during locomotion ,
muscle contraction induces an elevation 
 in intramuscular pressure and compresses the vascular bed , leading to cessation of 
 perfusion8 .
niizeki reported evidence of 
 a physiological cls using computer - assisted bilateral thigh - cuff occlusion to simulate 
 intramuscular pressure changes during bipedal locomotion2 . alternating cuff occlusion at hr resulted in phase synchronization , 
 and individual heart beats were unlikely to overlap with elevated cuff pressure . in 
 addition , kimura et al .
suggested that cls with an appropriate phase difference attenuates 
 peripheral vascular resistance and systolic blood pressure while increasing stroke 
 volume9 .
it is possible that there is a 
 specific phase difference between cardiac systole and muscle contraction that confers peak 
 leg arterial pressure during the lowest muscle capillary resistance .
first , the effect of respiratory 
 rhythm on the relationship between cardiac and locomotor rhythms was not assessed . 

respiration could indirectly influence the occurrence of cls as respiratory sinus arrhythmia 
 modulates the heartbeat through neural effects10 , and ventilation modulates venous return through movement of the 
 diaphragm11 .
second , changes in total 
 hb on the time scale of individual cls events could not be assessed . as a result , the 
 beat - by - beat cls physiological response during exercise was unclear .
additional studies are required to assess this 
 relationship in other populations , such as patients with cardiovascular diseases who may 
 benefit from cls to improve the capacity for therapeutic exercise .
 [ purpose ] the purpose of this study was to investigate whether the occurrence of 
 cardiac - locomotor synchronization ( cls ) improves lower leg muscle blood perfusion during 
 walking . 
 [ subjects and methods ] eleven healthy men were studied while performing two 
 treadmill protocols . 
 the cls protocol involved subjects walking at the frequency of their 
 heart rate ( hr ) to induce cls . the free protocol ( reference ) involved subjects walking at 
 a self - selected cadence . 
 the treadmill load was identical in the two protocols . 
 
 electrocardiographic signals for hr , foot switch signals for step rate and near - infrared 
 spectroscopy ( nirs ) signals for total haemoglobin ( total hb ) in the lower leg muscles were 
 measured continuously for 10  min after hr reached a steady state . 
 [ results ] the mean hr 
 and mean step rate did not differ between the cls and free protocols 
 . however , total hb 
 was significantly higher in the cls protocol than in the free protocol . 
 the rate of 
 increase in total hb positively correlated with the strength of cls . 
 [ conclusion ] these 
 results suggest that the occurrence of cls enhances lower leg muscle blood perfusion by 
 increasing the strength of cls during walking . 
INTRODUCTION
SUBJECTS AND METHODS
RESULTS
DISCUSSION