Plasma volume (PV) expansion through aerobic training has been suggested to improve heat dissipation mechanisms in young people (1–4), but the PV expansion response appears to be attenuated in older subjects (5–7). However, it remains unclear whether the PV response in older people is affected by walking training for several months, longer than that in previous studies (5,6,8), due to long-term exercise adaptation mechanisms.
On the other hand, supplementation with a mixture of carbohydrate (CHO) + whey protein during aerobic training at 60% to 70% of individual peak aerobic capacity (V˙O2peak), more than 3 d·wk−1, for several weeks, reportedly enhanced PV expansion with increased albumin content (Albcont) by causing fluid movement from the interstitial space to the plasma space according to the colloid osmotic pressure gradient to increase PV in older people (5,6,8). However, these results were obtained from subjects who performed aerobic training using cycle ergometers in a gym, and no studies have been conducted to examine whether the same effects were obtained by home-based walking training which can be performed easily by middle-age and older people.
One reason that no studies have examined these issues mentioned above is the lack of a tracking system to assess whether exercise intensity, duration, and frequency for a given period reach the level required to increase PV during home-based walking training for several months in older people. In the present study, we used the system that we developed to determine V˙O2peak for walking and to monitor exercise intensity during interval walking training (IWT), repeating a set of fast walking at >70% V˙O2peak and slow walking at approximately 40% V˙O2peak for 3 min each, more than five sets per day, >4 d·wk−1, and to transfer the data to a server computer through the internet (9,10). Using this system, we reported that IWT for 5 months increased thigh muscle strength by >10% and V˙O2peak by approximately 10% (11), with improved symptoms of lifestyle-related diseases (12). However, it is not clear whether PV and Albcont are increased by IWT and whether the responses are enhanced by a mixture of CHO and whey protein supplementation.
Therefore in study 1, we examined the hypothesis whether the baseline PV and plasma albumin content (Albcont) in individuals were associated with the number of IWT days for 12 months preceding the measurement. In study 2, we examined, in the same subjects as in study 1, the hypothesis whether a mixture of CHO + whey protein supplementation during an additional 5-month IWT would enhance increases in PV and Albcont. Furthermore, because fasting blood glucose concentration ([Glc]f) in the baseline is known to reflect insulin sensitivity in the peripheral tissue (13) and because improved insulin sensitivity during a long-term exercise adaptation would be involved in PV expansion through improved albumin synthesis in the liver (14,15), we examined the hypothesis whether there were any association of [Glc]f with PV and Albcont, in the baselines for study 1 and also with changes in PV (ΔPV) and Albcont (ΔAlbcont) after the additional 5-month IWT for study 2.
If we obtain results that support the hypotheses, the regimen may be broadly accepted by middle-age and older people who wish to increase PV to improve heat dissipation mechanisms (5–7) and prevent heat illness, of which the incidence is reportedly higher in middle-age and older people than in young people during midsummer in Japan (16).
Subjects and Grouping
Figure 1 shows a timeline of the present study. The procedure in this study was approved by the Institutional Review Board for Human Experiments, Shinshu University School of Medicine. The subjects were recruited from those who had performed IWT for more than 24 months in the “Jukunen Taiikudaigaku” project, which is a health promotion program for middle-age and older people in Matsumoto City. For the recruitment, we displayed a poster in a local community office that the participants visit regularly and distributed leaflets. After the experimental protocol was fully explained, 27 of 30 responders provided written informed consent and enrolled in the study. After the preintervention (PRE) measurement to determine the baseline values, we randomly divided the subjects (17 men and 10 women; age, 57–77 yr) into two groups: CHO (8 men and 5 women) and Pro-CHO (9 men and 5 women), consuming either glucose alone or a mixture of CHO + whey protein during IWT for the next 5 months, respectively. The postintervention (POST) measurement was performed after completion of the additional 5-month IWT (Table 1).
For the PRE measurement, the subjects were invited to a laboratory at 8:00 AM on the day assigned to individuals from May to June 2009 after an 11-h fasting period with free access to water. After conducting interviews on current health status, we measured height, body weight, arterial blood pressure, blood constituents, and PV. On another day within several days after the measurement, the subjects were invited to a gym to measure their peak aerobic capacity (V˙O2peak) for walking as described in detail below. The same variables were measured again in the POST measurement within several days after the additional 5-month IWT. In study 1, we analyzed the variables in the baselines relative to IWT days for 12 months before starting the additional 5-month IWT in individual subjects cross-sectionally. In study 2, we examined the effects of supplementation during the additional 5-month IWT on the variables longitudinally.
Interval Walking Training
Subjects were instructed to repeat more than five sets of fast (>70% V˙O2peak) and slow (~40% V˙O2peak) walking for 3 min each, more than 4 d·wk−1. Training intensity was monitored with a triaxial accelerometer (JD Mate; Kissei Comtec, Matsumoto, Japan) carried on the midclavicular line of the right or left waist. A beeping signal alerted subjects when a change of intensity was scheduled and another melody notified them when their walking intensity had reached the target level every minute. Every 2 wk, the subjects visited a local community office and the walking record from the tracking devices was transferred to a central server at the administrative center through the internet for automatic analysis and reporting. Trainers used the reports on exercise intensity and other parameters (Table 2) to instruct the subjects how best to achieve the target levels. The additional 5-month IWT in study 2 was performed between May 16, 2009, and November 4, 2009, during which period the daily average atmospheric temperature (Ta) ranged from 3.2°C to 27.9°C, and the relative humidity (RH) varied between 38% and 91%.
The CHO supplement (90 kcal) was composed of 22.5 g glucose, 0 g protein, 0 g fat, and 43 mg sodium (Weider in jelly multivitamin, Morinaga, Tokyo, Japan). The CHO + whey-protein supplement (100 kcal) was composed of 10 g whey protein and 15 g CHO mixture of fructose, dextrin, glucose, and maltose with 0 g fat and 10 to 30 mg sodium (JogMate, Otsuka, Tokyo). We adopt the supplement since we reported that the supplement intake after daily exercise during a 2-month cycling training increased PV and Albcont in older people (5,6).
Subjects in both groups were instructed to maintain their dietary habits, except for the supplements, during study 2. In addition, they were instructed to consume the assigned supplement within 30 min after daily exercise since albumin synthesis in the liver was enhanced during the period (17), but they were not allowed to consume any other foods or fluids except for water more than 60 min before and after exercise each training day. They were also instructed to report foods consumed for seven consecutive days between May and July 2009 using a questionnaire. A dietitian calculated the daily nutrition intake with commercially available software (Excel Eiyokun, FFqg, Ver 3.0; Kenpakusya, Co. Ltd., Tokyo, Japan). As a result, without the supplements, 12 subjects in the CHO group and 13 subjects in the Pro-CHO group consumed 1983 ± 145 and 1849 ± 119 kcal with diet; 252 ± 10 and 253 ± 15 g CHO, 77 ± 6 and 67 ± 5 g protein, 66 ± 7 and 56 ± 4 g fat, and 4626 ± 520 and 3812 ± 453 mg sodium per day, respectively, with no significant differences between the groups (P > 0.24), which met the recommended dietary allowances for active, older Japanese adults, except for a relatively high sodium intake (18).
V˙O2peak and V˙O2 during IWT measurements with calorimeter
We determined V˙O2peak by measuring energy expenditure with the accelerometer during graded intensity walking on a flat floor at a slow, moderate, and fast pace for 3 min each, as reported previously (12). This approach was reported to show good agreement with a graded cycling test (19). Also, we determined the oxygen consumption rate (V˙O2) during IWT using the calorimeter. Since the meter is equipped with not only a triaxial-accelerometer but also a barometer, we can estimate energy expenditure precisely even when subjects walk on an incline using a logic that we developed (19).
Arterial blood pressures at rest
We measured the systolic blood pressure (SBP) and diastolic blood pressure (DBP) of the subjects using the auscultation method from the right upper arm at the heart level by inflation of the cuff with sonometric pickup of Korotkoff’s sound (model STBP-780; Colin, Komaki, Japan) after sitting at rest more than 10 min in a room controlled to a Ta of approximately 28°C and RH of approximately 50%.
PV and blood constituents
On the day before the day of PRE and POST measurements, subjects were asked to eat a standardized breakfast and lunch at 7:00 AM and 12:00 noon, respectively, and to finish the standardized dinner by 9:00 PM. Food was controlled over the course of the day (i.e., standardized breakfast, lunch, and dinner): total calories were approximately 2100 kcal, total CHO were approximately 330 g, total protein approximately 67 g, total fat approximately 56 g, and sodium approximately 2.4 g before and after training in the CHO and Pro-CHO groups. The subjects reported to the laboratory at 8:00 AM normally hydrated but without having eaten any food for at least approximately 11 h before the measurement. To ensure that they were appropriately hydrated, they were asked to drink approximately 500 mL water 2 h before the visit. After emptying their bladders, they were weighed and entered a room controlled to a Ta of approximately 28°C and RH of approximately 50%. An 18-gauge Teflon catheter was then placed in the right antecubital vein for blood sampling and Evans blue dye injection. After the subjects rested in a sitting position for 30 min, the PV was determined using the Evans blue dye dilution method (7,20). Briefly, baseline blood samples were taken, the dye was injected, the blood samples were taken at 10 min after injection, and the absorbance (620 and 740 nm, U-1500; Hitachi, Tokyo) was used to determine PV.
An aliquot of the baseline blood sample was transferred to a heparin-treated tube and used to determine hematocrit (Hct, microcentrifuge), hemoglobin concentration ([Hb], sodium lauryl sulfate hemoglobin method; Sigma Chemical, St Louis, MO) in triplicate, and hemoglobin A1c (HbA1c, DM-Jack II; Kyowa Medex, Tokyo, Japan). The remaining aliquot of the sample was transferred to a heparin-treated tube and centrifuged at 4°C for 30 min. The separated plasma was used to determine the total plasma protein ([TP]) by refractometry, plasma albumin concentrations ([Alb]) by the bromcresol green method (Wako Chemical, Tokyo), and [Glc]f (YSI 2300 Stat Plus; Yellow Springs, OH). Albcont was calculated as a product of PV and [Alb].
We analyzed PV, Albcont, and [Glc]f in the PRE measurement relative to the number of IWT days for 12 months, April 2008 to March 2009, using 17 male (M) and 10 female (F) subjects before starting the additional 5-month IWT for study 2.
We analyzed any significantly different effects of the supplements, CHO or Pro-CHO, during the additional 5-month IWT from May to November 2009, on the variables by comparing the PRE and POST measurements between the groups using 7 M and 5 F subjects in the CHO group and 9 M and 5 F subjects in the Pro-CHO group, because one M subject was absent from the POST measurement (Fig. 1).
A two-way (one-between supplements [CHO vs Pro-CHO] and one-within time (pre vs post additional 5-month IWT) ANOVA for repeated measures was used to examine any significant differences in variables (Table 1). A one-way ANOVA was used to examine the effects of supplements (CHO vs Pro-CHO) on the training achievement (Table 2). The standard regression analysis was used to analyze the relationships between IWT days versus PV and Albcont (Fig. 2A, B) and between Albcont versus PV (Fig. 2C) in study 1. The analysis was also used to determine the relationships between [Glc]f versus PV and Albcont after pooling the values in the PRE and POST measurements (Figure 1A and B, Supplemental Digital Content, Relationships between fasting plasma glucose concentration ([Glc]f) versus PV [A] and plasma albumin content (Abcont) [B] in 16 M and 10 F subjects before and after additional 5-month IWT for study 2, http://links.lww.com/MSS/B33) and also between the baseline [Glc]f versus changes in PV (ΔPV), Albcont (ΔAlbcont), and HbA1c (ΔHbA1c) after the additional 5-month IWT in study 2 (Fig. 3A, B, C). When we examined the effects of supplements (CHO vs Pro-CHO) on ΔPV, ΔAlbcont, and ΔHbA1c after the additional 5-month IWT in study 2, we corrected the changes using ANCOVA with the baseline [Glc]f as a covariate (Fig. 4A, B, C) after confirming that the [Glc]f significantly affected ΔPV, ΔAlbcont, and ΔHbA1c (Fig. 3A, B, C). Similarly, we determined the relationship between ΔAlbcont and ΔPV after the correction (Fig. 3D). The statistical power (1 − β) is presented in the text at α = 0.05 when the variables were significantly different between the CHO and Pro-CHO groups. All values are expressed as means ± SE. The null hypothesis was rejected at P < 0.05.
Table 1 shows the physical characteristics, blood pressures, and plasma constituents of the subjects in studies 1 and 2. The values in study 1 were from the PRE measurement in all subjects, and the values in study 2 were presented from the PRE and POST measurements in the CHO and Pro-CHO groups, respectively. We observed a significant increase of HbA1c only in the CHO group (PRE vs POST, P = 0.004). There were no significant differences in other variables—age, height, body mass index (BMI), V˙O2peak, HRpeak, SBP, DBP, PV, [TP], [Alb], and [Glc]f—before and after the additional 5-month IWT in each group or between groups (all, P > 0.08), with no interactive effects of (supplements [CHO vs Pro-CHO] × Time [PRE vs POST measurement]) on the variables (all, P > 0.07).
As shown in Figure 2, there was a positive correlation between the baseline PV versus the number of training days for 12 months preceding the PRE measurement (M: r = 0.670, P = 0.006, F: r = 0.808, P = 0.005, total: r = 0.716, P < 0.001) [A] and Albcont (M: r = 0.509, P = 0.053, F: r = 0.882, P = 0.001, total: r = 0.671 P < 0.001) [B] with a high correlation between PV (y) and Albcont (x) in all subjects with a regression equation of y = 21.5 x + 2.5 (r = 0.930, P < 0.001) [C]. On the other hand, there were no significant correlations between the baseline [Glc]f or HbA1c versus the number of training days for 12 months (both, P > 0.2).
In addition, the baseline [Glc]f was negatively correlated with the baseline PV (r = −0.445, P = 0.020) and Albcont (r = −0.393, P = 0.042) in the PRE measurement. When the values in the PRE and POST measurements were pooled, [Glc]f was also negatively correlated with PV (r = −0.460, P = 0.001) and Albcont (r = −0.384, P = 0.004) [see Figure, Supplemental Digital Content, Relationships between fasting plasma glucose concentration ([Glc]f) versus PV [A] and plasma albumin content (Abcont) [B] in 16 M and 10 F subjects before and after additional 5-month IWT for study 2, http://links.lww.com/MSS/B33]. The open symbols indicate the subjects in the CHO group and the closed symbols indicate those in the Pro-CHO group. The squares indicate M subjects, and the circles indicate F subjects. No other variables in Table 1 were significantly correlated with PV and Albcont (all, P > 0.1).
Table 2 shows IWT achievements for 12 months preceding the PRE measurements in study 1 and the achievements of the CHO and Pro-CHO groups in study 2. As shown in the table, the number of IWT days per week in both groups for study 2 was significantly higher than those in study 1 (both, P < 0.01), but there were no significant differences in these variables between the groups (all, P > 0.443).
As in Figure 3, the baseline [Glc]f in the PRE measurement was significantly correlated with ΔPV [A], ΔAlbcont [B], and ΔHbA1c [C] after the additional 5-month IWT in study 2. Therefore, we determined the relationship between ΔPV and ΔAlbcont after correcting for the [Glc]f by ANCOVA. We found that ΔPV (y) was highly correlated with ΔAlbcont (x) with a regression equation of y = 22.5 x − 0.1 (r = −0.960, P < 0.001) [D].
As in Figure 4A, B, we found that PV and Albcont corrected for the [Glc]f by ANCOVA marginally decreased from the baselines in the CHO group (P = 0.081 and P = 0.130, respectively), but they remained unchanged in the Pro-CHO group, with significant differences in the changes between groups (P = 0.020, 1 − β = 0.621 and P = 0.041, 1 − β = 0.512, respectively). In addition, we found that ΔHbA1c after correcting for the [Glc]f by ANCOVA increased in the CHO group (P < 0.001) and remained unchanged in the Pro-CHO group with a significant difference between groups (P = 0.018, 1 − β = 0.687) (Fig. 4C).
In the present study, we found that the baseline PV and Albcont were proportional to the number of IWT days for 12 months preceding the PRE measurement, and they were negatively correlated with the baseline [Glc]f. In addition, we found that CHO + whey protein supplementation during the additional 5-month IWT prevented the marginal reductions in PV and Albcont and the increase in HbA1c in the CNT group for study 2. These changes were significantly different between groups after correction for the baseline [Glc]f by ANCOVA.
The BMI, height, and V˙O2peak values reported in this study (Table 1) were similar to those previously reported in age-matched Japanese populations (12,21,22), but V˙O2peak was slightly higher in this study population than those populations, probably because our subjects had performed IWT for more than 24 months before participating in the present study. Therefore, the characteristics of the subjects in this study generally reflected those of this age group of the Japanese population.
PV expansion vs IWT achievements for 12 months
In contrast to the results in the previous studies (5–7) reporting that aerobic training using the cycle ergometer for 8 and 18 wk caused no PV expansion in older subjects, we found in the present study that the baseline PV and Albcont were proportional to the number of IWT days for 12 months preceding the PRE measurement (Fig. 2A, B) with a high correlation between PV and Albcont (Fig. 2C). In addition, we found that [Glc]f was inversely correlated with PV and Albcont in the PRE measurement, and moreover, this was confirmed when the values in the PRE and POST measurements were pooled (Fig. 1A and B, Supplemental Digital Content, Relationships between fasting plasma glucose concentration ([Glc]f) vs PV [A] and plasma albumin content (Abcont) [B] in 16 M and 10 F subjects before and after additional 5-month IWT for study 2, http://links.lww.com/MSS/B33). These results suggest that IWT for several months, longer than the several weeks used in previous studies (5,6), increases PV and that glucose metabolism is involved in this response.
The detailed association of PV and Albcont with [Glc]f in the PRE measurement remained unknown; however, it has been suggested that the higher [Glc]f is suggestive of the lower insulin sensitivity in the peripheral tissues (13). Because insulin is known to stimulate the albumin synthesis rate in the liver (14,15), the lower sensitivity in subjects with higher [Glc]f might attenuate PV expansion by aerobic training. As [Glc]f likely decreases with an increased number of IWT days—although we found no direct and significant correlations between them most likely because of the high inter-individual variation in [Glc]f—the significant correlations between the number of IWT days versus PV and Albcont in Figure 2 might be associated with improved glucose metabolism attained by IWT for several months. This idea might be supported by the results in study 2.
PV expansion by 5-month IWT with supplementation
We conducted study 2 to examine the effects of CHO + whey protein supplementation immediately after daily IWT for the additional 5 months. Five months was chosen for IWT because the improvements in physical fitness and in the symptoms of lifestyle-related diseases, including [Glc]f and HbA1c, appeared after this period (11,12). As a result, there were no significant differences in ΔPV, ΔAlbcont, except for the increase in HbA1c on the CHO group (Table 1); however, when corrected for the baseline [Glc]f by ANCOVA after confirming that the [Glc]f was significantly correlated with the changes (Fig. 3), we found that PV and Albcont marginally decreased with an increase in HbA1c in the CHO group, whereas they remained unchanged in the Pro-CHO group. We also found that ΔPV was highly correlated with ΔAlbcont with a similar regression coefficient to that in Figure 2C (Fig. 3D). Significant differences in ΔPV, ΔAlbcont, and ΔHbA1c were evident between the two groups (Fig. 4), whereas we failed to detect any significant difference in Δ[Glc]f, probably due to high interindividual variation. These results suggest a close association between PV expansion and glucose metabolism through increased Albcont in older subjects.
As for the possible association between PV and glucose metabolism, Masuki et al. (23) recently examined the effects of milk product supplementation during IWT for 5 months in older women who had performed IWT for more than 6 months before participating in the study, almost equivalent to the Pro-CHO group in the present study, and suggested that activity (methylation) of proinflammatory cytokine genes, such as NFKB1 and NFKB2, in leukocyte were suppressed (enhanced) by the supplementation. Because chronic inflammation with aging has been suggested to be one of the major causes of lifestyle-related diseases including diabetes mellitus (24), no reduction in PV and Albcont in the Pro-CHO group in Figure 4 might be at least in part caused by the suppression of chronic inflammatory responses to enhance insulin sensitivity in the liver.
The detailed mechanisms for the marginal decreases in PV and Albcont and the significant increase in HbA1c after the additional 5-month IWT in study 2 for the CHO group (Fig. 4) remain unknown; however, seasonal variation of PV, decreasing in winter and increasing in summer (25), and seasonal variation of [Glc]f; increasing in winter and decreasing in summer (26) have been suggested. On the other hand, Nakano et al. (27) recently suggested seasonal variation of proinflammatory gene (NFKB2) activity; most inactivated (most methylated) in April to June and most activated (most demethylated) in October to November. In addition, Masuki et al. (23) suggested in the above study that NFKB1 and NFKB2 genes and other related inflammatory genes in the group with no milk product supplementation, equivalent to the CHO group in the present study, were marginally activated (demethylated) after the 5-month IWT from April to September, similar to the period in the present study. These results suggest that the marginal reductions in PV and Albcont for the CHO group (Fig. 4A, B) might link with the seasonal variation of [Glc]f which can be partially explained by seasonal fluctuation of chronic inflammation levels in the body.
As shown in Figure 2, PV and Albcont were proportional to the IWT days for 12 months in study 1 but both marginally decreased after 5 months of IWT in the CHO group for study 2 as shown in Figure 4. The reasons for the discrepancy at a glance might be that the increases in PV and Albcont attained by IWT alone had likely reached plateau levels by more than 24-month training, but the levels might be influenced by seasonal fluctuation of glucose metabolism with a chronic inflammation state in the body for some reason. In the present study, we found that a mixture of CHO and whey protein supplementation during IWT prevented these responses.
In the present study, we did not measure thermoregulatory responses. However, Okazaki et al. (5) examined the effects of a mixture of CHO and whey protein intake on PV and thermoregulatory responses during exercise in a hot environment in older men and suggested that the supplement increased PV by approximately 6% to improve the sweat rate response by 18% and the cutaneous vasodilator response by 89% at a given increase in esophageal temperature. Recently, Kataoka et al. (6) examined the effects of the supplement in hypertensive older men and reported similar results with reduced arterial blood pressure. In the present study, because the difference in the change of PV after the additional 6-month IWT between the CHO and Pro-CHO groups was similar as in previous studies (5,6), the thermoregulatory responses would be improved in the Pro-CHO group compared with the CHO group as in the previous studies (5,6).
PV and Albcont were proportional to the number of IWT days for 12 months, and a mixture of CHO + whey protein supplementation during IWT for the additional 5 months prevented the marginal reduction in PV and Albcont in the supplementation of CHO alone, which might be partially linked with blood glucose control mechanisms.
This study was supported by grants from the Japan Society for the Promotion of Science (21650168 & 24240089). The authors thank all the volunteers for their participation in this study.
No conflicts of interest, financial or otherwise, are declared by the authors. The results of the present study do not constitute endorsement by the American College of Sports Medicine. The results of the study are presented clearly, honestly, and without fabrication, falsification, or inappropriate data manipulation.
The present address for Y. Kamijo is Department of Rehabilitation Medicine, Wakayama Medical University, 811-1 Kimiidera, Wakayama 641-8509, Japan.
1. Goto M, Okazaki K, Kamijo Y, et al. Protein and carbohydrate supplementation during 5-day aerobic training enhanced plasma volume expansion and thermoregulatory adaptation in young men. J Appl Physiol (1985)
2. Ichinose T, Okazaki K, Masuki S, et al. Ten-day endurance training attenuates the hyperosmotic suppression of cutaneous vasodilation during exercise but not sweating. J Appl Physiol (1985)
3. Ikegawa S, Kamijo Y, Okazaki K, Masuki S, Okada Y, Nose H. Effects of hypohydration on thermoregulation during exercise before and after 5-day aerobic training in a warm environment in young men. J Appl Physiol (1985)
4. Takeno Y, Kamijo YI, Nose H. Thermoregulatory and aerobic changes after endurance training in a hypobaric hypoxic and warm environment. J Appl Physiol (1985)
5. Okazaki K, Ichinose T, Mitono H, et al. Impact of protein and carbohydrate supplementation on plasma volume expansion and thermoregulatory adaptation by aerobic training in older men. J Appl Physiol (1985)
6. Kataoka Y, Kamijo YI, Ogawa Y, et al. Effects of hypervolemia by protein and glucose supplementation during aerobic training on thermal and arterial pressure regulations in hypertensive older men. J Appl Physiol (1985)
7. Okazaki K, Kamijo Y, Takeno Y, Okumoto T, Masuki S, Nose H. Effects of exercise training on thermoregulatory responses and blood volume in older men. J Appl Physiol (1985)
8. Okazaki K, Hayase H, Ichinose T, Mitono H, Doi T, Nose H. Protein and carbohydrate supplementation after exercise increases plasma volume and albumin content in older and young men. J Appl Physiol (1985)
9. Nose H, Morikawa M, Yamazaki T, et al. Beyond epidemiology: field studies and the physiology laboratory as the whole world. J Physiol
. 2009;587(Pt 23):5569–75.
10. Masuki S, Morikawa M, Nose H. Interval walking training can increase physical fitness in middle-aged and older people
. Exerc Sport Sci Rev
11. Nemoto K, Gen-no H, Masuki S, Okazaki K, Nose H. Effects of high-intensity interval walking training on physical fitness and blood pressure in middle-aged and older people
. Mayo Clin Proc
12. Morikawa M, Okazaki K, Masuki S, et al. Physical fitness and indices of lifestyle-related diseases before and after interval walking training in middle-aged and older males and females. Br J Sports Med
13. McCarthy ST, Harris E, Turner RC. Glucose control of basal insulin secretion in diabetes. Diabetologia
14. De Feo P, Gaisano MG, Haymond MW. Differential effects of insulin deficiency on albumin and fibrinogen synthesis in humans. J Clin Invest
15. Ahlman B, Charlton M, Fu A, Berg C, O’Brien P, Nair KS. Insulin’s effect on synthesis rates of liver proteins. A swine model comparing various precursors of protein synthesis. Diabetes
16. Nakai S. [Recent and past incidence of heat illness in Japan]. J Public Health Practice
17. Sheffield-Moore M, Yeckel CW, Volpi E, et al. Postexercise protein metabolism in older and younger men following moderate-intensity aerobic exercise. Am J Physiol Endocrinol Metab
18. Ministry of Health L, and Welfare of Japan. Recommended dietary allowances. In: [Dietary Reference Intakes for Japanese 2010
]. (2nd ed) [ in Japanese]. Tokyo, Japan: Daiichi Shuppan; 2010. pp. 43–117, 89–91.
19. Yamazaki T, Gen-No H, Kamijo Y, Okazaki K, Masuki S, Nose H. A new device to estimate VO2 during incline walking by accelerometry and barometry. Med Sci Sports Exerc
20. Greenleaf JE, Convertino VA, Mangseth GR. Plasma volume during stress in man: osmolality and red cell volume. J Appl Physiol Respir Environ Exerc Physiol
21. Niizeki T, Takeishi Y, Takabatake N, et al. Circulating levels of heart-type fatty acid-binding protein in a general Japanese population: effects of age, gender, and physiologic characteristics. Circ J
22. Laboratory of Physical Fitness Standards. Body Mass Index, Maximum Oxygen Intake. In: [New Physical Fitness Standards of Japanese People
]. 2nd ed. Tokyo, Japan: Fumaido Shuppan; 2007. pp. 1–421.
23. Masuki S, Nishida K, Hashimoto S, et al. Effects of milk product intake on thigh muscle strength and NFKB gene methylation during home-based interval walking training in older women: a randomized, controlled pilot study. PLoS One
24. Handschin C, Spiegelman BM. The role of exercise and PGC1alpha in inflammation and chronic disease. Nature
25. Doupe J, Ferguson MH, Hildes JA. Seasonal fluctuations in blood volume. Can J Biochem Physiol
26. Suarez L, Barrett-Connor E. Seasonal variation in fasting plasma glucose levels in man. Diabetologia
27. Nakano S, Masuki S, Morikawa M, Takasugi S, Nose H. Effects of milk intake + 1-mo interval walking training on NFKB2
gene methylation in older men. J Physical Fit Sports Med