We selected 14 healthy volunteers (mean age, 41 ± 15.2 years, ranging 19 - 60 years old in both sexualities) and informed consented according to The Ethics Committee of Kanazawa Medical University. The contents of sexuality were 56.25% of the lady and 43.75% for gentleman. They were the students of Medical University and the stuff for the school of medicine. None of them was a specialist for athletic field. The group was set up into two groups according to the intense of walking, 4 mets and 8 mets.

These subjects participated in this study after giving their informed consent. We conducted the walking exercise at the country side of Japan. The course of exercise located at 40 meter in high and 1015 mb of atmospheric pressure. We sampled peripheral blood from the 14 volunteers before and after exercise, at the same time on each day, in consideration of circadian rhythm of leukocyte. The exercise start at ten o’clock for about one hour, corresponding 4 metabolic rates; mets [21-24]. Time interval of blood sampling between before and after exer-

cise was approximately 24 hours. Measurements of the total leukocyte were assessed for differential leukocyte counts and granulocyte and lymphocyte ratio in the peripheral blood We ordered to the laboratory of Ishikawa Prefecture Preventive Medicine Association for precise counts for the total and differential leukocyte counts in the peripheral blood from the subjects. The total and differential leukocyte counts were measured by the automated hematology analyzer XE-2100 (Sysmex, Inc., Kobe, Japan).

After two weeks of cooling down for all volunteers, we set up again for the same exercise except for the intense of walking was 8 km/hour for the same kilo-meter of the same course (8 mets). We prepared peripheral blood from the same 14 volunteers before and after the exercise, at the same time on each day, in consideration of circadian rhythm of leukocyte. We conducted the walking exercise at the same course in country side of Japan (Figures 1 and 2).

For the purpose of estimating CD positive cell, the whole blood obtained from the subjects by blood collection tube containing an anticoagulant EDTA-2K. 100 μl of whole blood were added the antibody. After washing with PBS, the suspensions were mixed phycoerythrin (PE)-conjugated streptavidin (Beckman Coulter Inc. France) and fluorescence monoclonal antibody: peridinin chlorophyll protein-cyanin 5.5 (PerCP-Cy5.5)-conjugated CD2, fluorescein isothiocyanate (FITC)-conjugated CD4, FITC-conjugated CD8, FITC-conjugated CD16, FITC-conjugated CD19, FITC-conjugated CD57 (each Becton Dickinson Co. U.S.A.), allophycocyanin (APC)-conjugated CD8, and APC-conjugated CD57

(each Beckman Coulter). The negative controls were added PE-conjugated streptavidin and the isotype control antibodies to the CD antibodies. After incubation for 30 minutes at 4˚C, these samples were hemolyzed using a 10-times dilution FACS Lysing Solution (Becton Dickinson). After washing with PBS, the cell suspensions were fixed using a 10-times dilution CellFIX (Becton Dickinson) and analyzed by flow cytometer FACS Caliber (Becton Dickinson) [18-20].

The statistical comparisons between two groups (before and after walking) for the test of significant difference were performed using paired t-test and wilcoxon signed-ranks test. Further, the test of the correlation were performed a spearman’s correlation coefficient by rank test. Data are expressed as means ± standard error of mean (SE). A P value < 0.05 was considered to be statistically significant. The Kendall tau Rank Correlation and the two-sided p-value. The ordinary scatter plot and the scatter plot between ranks of X axis & Y axis are also shown.

Each volunteer was prepared their blood before start for exercise and after informed consented to the experimental purpose by written Ethics of the Committee in Kanazawa Medical University. After warming up for 5 minutes by Radio exercise by NHK. We suggested to take a walk for one hour for 4 km of the curse (4 mets). We tried to exhibit the effect of peripheral total leukocyte number by individual level of change and plot in the x-axis as in each value before the exercise. As was in Figure 3, The relative value (%) was calculated before and after the exercise and plotted in the figure of the x-axis according to the value before exercise. As a result of the exhibition, there found three groups, separated, upregulated individuals and down-regulated one and was no change. The correlation of change was expressed as a linear function and significant reverse correlation, −0.6893 indicating ideal value of correlative index −0.5 (Figure 3). The data obtained from 4mets exercise was brought ideal regulation. However, 8mets could not bring such a effect significantly. About the leukocyte subset, the ideal regulation of lymphocyte and monocyte were found by 4 mets, but not in granulocyte.

After 2-weeks of cooling down, we set up the same mode of trial at the same course and by the same volunteers. None of volunteer was dropped out with any serious problem. In this time, we set up 8 mets impacts of exercise, asking to walk out by half an hour for the same course of 4 kilometers (8 mets). Preparing 5-min. warming up, we tried to express the effect of peripheral total leukocyte number by individual level of change and plot in the x-axis as in each value before the exercise as is in 3.1. (Figure 3). As shown in lower panel in Figure 3, The correlation was exhibited on a linear function but good correlation was retreated from the results by the case by 8 mets. The slant value was −0.3617. The data obtained from 8mets exercise was lesser regulatory effect than that compared by 4 mets of exercise. About the leukocyte subset, the regulation by lymphocytes were more remarkable. However, only of the monocyte exhibited significant change by both impact 4 and 8 mets.

We had been observed the reputational effect of exercise could be evidenced within a short period. But the possibility still remain that the leukocyte change was happen to emerge for the dairy life as accidental factor, such as stress and so on. In order to avoid such possibility, then we tried to show the change of the peripheral leukocyte number was the result of another network system of the inner system such as peripheral leukocyte, endocrine and brain system. We tried to access the effect CD positive lymphocyte. They were CD2, CD CD4, CD8, CD16, CD19, CD57. Within those CD positive cells, CD2, CD4, CD8, CD19 cells were regulated significantly as in number of each CD marker positive cells by 4 mets of exercise.

After 2 weeks of cooling down, we set up the same mode of trial at the same course and by the same volunteers. None of volunteer was dropped out with any serious problem. In this time, we set up 8 mets impacts of exercise, asking to walk out by half an hour for the same course of 4 kilometers (8 mets). Preparing 5-min warming up, we tried to express the effect on lymphocyte subsets as CD positive number. They were CD2, CD 4, CD 8,

CD 16, CD 19, CD 57. As shown in Figure 4, the regulatory effect was disappeared in the all the subset in 8 mets of exercise.

As shown in Figures 3 and 5, we can compared and got the result that of 4mets of exercise was more effective to regulate lymphocyte and lymphocyte subset by individual change by plotting the linear function. In Figures 4 and 6, we tried to exhibited the same data with grouped and tried to make mean. From the results, there was no significant change between the value before and after the exercise both with 4 and 8 mets.

Our results showed that within 24 hours after light exercise, the white blood cells in peripheral blood had regulated significantly, not only in leukocyte subset but also lymphocyte subset. The results showed that these subsets could reflect the number and function of immuno-competent cells [13-19]. For example, in an individual with a low granulocyte number, the number increased after treatment, while it decreased in another individual with a higher cell number. Our results led us to believe that leukocyte subsets could be an interesting indicator for the evaluation of alternative therapies [15-20] Many systems are in place to evaluate Western therapies that aim at healing the symptoms of an illness[25-27]. We hope that our work will attract more attention to the mechanisms of which each CAM menu regulates the human immune system. Abo reported that according to the lymphocyte subset content, lymphocyte rich type showed over 40% on the other hand granulocyte rich type show over 60% of granulocyte [28]. Each type exhibited different character even in the same age, sexuality and different age. Within the same age and the sexuality, even in gentleman can sorts out as G-rich type (granulocyte 60%), and L-rich type (lymphocyte 40%). On the other hand, as a stand point of sexuality difference, the lady belongs to L-rich type but the gentleman belongs G-rich type. According to the age-related change, G-rich type of young man change to L-rich type according to getting older [29,30].

We have been trying to regulate the immune responsiveness through much mature by fragile daily condition from circumstance stress and so on. The main menu were, acupuncture, hot-spring hydrotherapy, light exercise etc. In this article, we would like to show the regulatory mechanism of the light exercise walking as a tailored scale. The circumstance, except for cases of contraindication, has been medically useful approved to be effective in many stress-related disorders and the improvement of dysfunction of the biological rhythm disturbance as well as chronic disease. The mechanism of effects has been reported in many studies, but many things are still unclear.

In order to assess correctly to the changes after the menu

menu, it is important to conscious of circadian rhythm. Abo also reported that it was possible to sort the constitution, granulocyte-rich individual and lymphocyte-rich one with the peripheral leukocyte [31,32].

Each population of subset is depends on a circadian rhythm. Within a same individual, granulocyte increase in the daytime, on the other hand, lymphocyte increased in the night time in a cycle 24 hrs. So we have to compare the effect of each menu for the peripheral leukocyte on the same time before and after the menu.

With our report simple comparison of grouped value did not exhibit the valuable change in each individual. So this style of presentation was suggestive to the patient for immunodeficy such as in DM.

It was reported that the leukocyte subset, granulocyte and lymphocyte regulated by various factors. One major point is that they are regulated by autonomous nervous system, resulting in circadian rhythm [30-33]. Therefore, in order to access the effect within a short time, it is necessary to consider this factor to adjust the time to collect the sample. For example, efficacy and impact of walking exercise has been widely recognized. However, the majority of walkers did not have a scientific background to know the best exercise menu for the one’ QOL; quality of life. The purpose of this study was to demonstrate the best menu of walking that regulates the peripheral white blood cells in number and function as a marker of QOL expression.

However, almost all the judgments of efficacy are VAS (visual analog scale). Moreover, simple processing by grouped value and make mean fadeout the precise regulation according to each constitution. Therefore, we hope our tailored scale can suggest assessing every CAM therapies in the world for competition (Figures 4 and 7).