Mechanisms of Ageing and Development
115 (2000) 61 – 71
www.elsevier.com/locate/mechagedev

Influence of short-term repeated fasting on the
longevity of female (NZB×NZW)F1 mice
Hiroshi Sogawa *, Chiharu Kubo
Department of Psychosomatic Medicine, Graduate School of Medical Sciences, Kyushu Uni6ersity,
3 -1 -1 Maidashi, Higashi-ku, Fukuoka, 812 -8582, Japan
Received 25 October 1999; received in revised form 26 February 2000; accepted 28 February 2000

Abstract
Caloric restriction in rodents is well known to retard the rate of aging, increase mean and
maximum life-spans, and inhibit the occurrence of many age-associated diseases. However,
little is known about the influence of short-term repeated fasting on longevity. In this study,
female (NZB×NZW)F1 mice were used to test the physiological effect of short-term
repeated fasting (4 consecutive days, every 2 weeks). The results showed that fasting mice
survived significantly longer than the full-fed mice, in spite of the fasting group having a
heavier body weight than the control group. Mean survival times for fasting and control
mice were 64.0 915.3 and 47.9 99.4 weeks, respectively. Short-term repeated fasting manipulation was also effective on the prolongation of life-span in autoimmune-prone mice.
© 2000 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Caloric restriction; Fasting; Autoimmune; Longevity

1. Introduction
Caloric restriction prolongs the mean and maximum life spans and inhibits
age-related diseases in rodents (McCay et al., 1935; Fernandes et al., 1978; Kubo et
al., 1984a,b, 1987, 1992a,b; Weindruch et al., 1986; Masoro, 1988; Weindruch and
Sohal, 1997). Many experiments using this manipulation have been done to test the
key concept of ‘undernutrition without malnutrition’. Although a number of
* Corresponding author. Tel.: +81-92-6425323; fax: + 81-92-6425336.
E-mail address: sogawa@cephal.med.kyushu-u.ac.jp (H. Sogawa)
0047-6374/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved.
PII: S 0 0 4 7 - 6 3 7 4 ( 0 0 ) 0 0 1 0 9 - 3

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hypotheses have been proposed, the precise mechanism responsible for this effect is
not definitely understood. Fasting therapy was first used mainly for the treatment
of obesity (Duncan et al., 1963; Drenick et al., 1964; Wing et al., 1983a), however,
it has also been notably effective in the treatment of psychosomatic disorders, and
is a common treatment for psychosomatic diseases in Japan (Yamamoto et al.,
1979). Our experience with patients with allergic or gastroenteric diseases indicates
that fasting therapy is very effective. To test our hypothesis that the effects of short
term repeated fasting might also influence longevity or disease in rodents, we did an
animal experiment using short-lived, autoimmunity-susceptible (NZB×NZW)F1
(B/W) mice, which represent one of several short-lived autoimmune-prone strains.
Mice of this strain have been extensively studied as a model of human lupus
erythematousus (Theofilopoulos and Dixon, 1986). These mice spontaneously develop autoimmune manifestations including formation of various autoantibodies
and also develop a fatal immune complex glomerulonephritis. Profound influences
of diet on development and expression of autoimmune disease in some strains of
mice have previously been reported (Kubo et al., 1984a,b, 1987, 1992a,b). In B/W
mice, life span has been doubled and sometimes even tripled by reduced caloric
intake (Kubo et al., 1987), however, little is known about the effects of repeated
fasting. In the present experiments, the effect of repeated fasting on body weight,
immunological function and longevity is presented.

2. Materials and methods

2.1. Mice
(NZB × NZW)F1 (B/W) mice were bred in our colony and weaned at 6 weeks of
age. Specific pathogen-free conditions were maintained throughout the period of
this study. The room was operated on a 12-h light/12-h dark cycle at constant
temperature and humidity. At 6 weeks of age, female mice were housed in metal
cages (five animals per cage) and randomly assigned to one of two groups, a
repeated fasting group or a control group fed ad libitum, each group consisting of
ten mice for the evaluation of survival, and five mice for the representative
immunological evaluation at 26 weeks of age. The mice were monitored until death
to establish relative survival times. Dead animals were removed when discovered
during daily checks of the cages.

2.2. Fasting regimen
Fasting periods were for 4 consecutive days, every 2 weeks. When fasting was
started, the fasting mice had access to only water. Except for these 4 days, the
animals had free access to food and water. The control animals were fed commercial lab diet (CLEA rodent diet, Osaka, Japan) ad libitum; the same as the fasting
group diet. The composition of the diet was as follows; 8.9% water, 25.4% soybean
meal as protein, 4.4% vegetable cooking oil as fat, 4.1% dehydrated alfalfa meal as

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63

fiber, 6.9% crude ash, 50.3% cereals as carbohydrates, minerals and various
vitamins. All animals were usually weighed just before the fasting period began.
This fasting protocol was begun at 6 weeks of age and continued to death.
Representative mice were sacrificed at 26 weeks of age to permit immunological
analysis for evaluation.

2.3. Proteinuria
Proteinuria was always assayed just before the fasting period with tetrabromphenol paper (Bayer-Sankyo, Tokyo, Japan ) on fresh urine samples. The test is graded
1–4+ (1+, 30 mg/100 ml; 2+, B100 mg/100 ml; 3+, B 300 mg/100 ml; and 4+,
B 1000 mg/100 ml). In this experiment, high-grade proteinuria was designated as
] 2+.

2.4. Cell preparation for immunologic assay
Mice were bled by cutting the femoral arteries and sacrificed by cervical dislocation. Spleens were collected aseptically. Single cell suspensions were made by gently
squeezing spleen tissue between two glass slides in Hanks’ balanced salt solution
(Gibco, Grand Island, NY), with gauze filtration to remove large residual fragments. Cells were washed three times with Hanks’ balanced salt solution before use.

2.5. Culture medium
Added to RPMI 1640 culture medium (Gibco) were 1 mM sodium pyruvate, 5
mM HEPES, penicillin (100 units/ml), 100 mg/ml of streptomycin, 5 × 10 − 5 mol of
2-mercaptoethanol, and 10% FCS. Normal CBA/H mouse serum (1%) was used
instead of FCS to assay responses to mitogen stimulation or mixed lymphocyte
reaction.

2.6. Mitogen stimulation
Mitogen-induced blastogenesis was measured using: phytohemagglutinin P
([PHA-P] Difco, Detroit, MI), 0.1% v/v; concanavalin A ([ConA] Calbiochem, La
Jolla, CA), 2 mg/ml; Salmonella typhosa lipopolysaccharide ([LPS] Difco), 50 mg/ml
in RPMI 1640 medium.

2.7. Natural killer (NK) cell acti6ity, mixed lymphocyte reaction (MLR)
Natural killer cell activity, and mixed lymphocyte reaction assay were measured
as previously described (Kubo et al., 1992b). Spleen cells of C57BL/6 mice were
used for stimulator cells of MLR.
This experiment was reviewed by the Ethics in Animal Experimentation Committee of the Faculty of Medicine, Kyushu University and carried out under the
control of the Guidelines for Animal Experiments of the Faculty of Medicine,

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Kyushu University and The Law (no. 105) and Notification (no. 6) of the Japanese
Government.

2.8. Statistics
Statistical analyses were performed with Student’s t-test for parametric data.
Survival rates were calculated by use of the Kaplan–Meier’s method. For all
analyses, the significance level was set at P= 0.05. Data analysis was done with
Statview Version 5.0 software (Abacus Concepts, Berkeley, CA, USA) on a
Macintosh computer.

3. Results

3.1. Growth cur6es
Mice were of two groups; a fasting group and a control group. The body weight
of each group before, during, and after the first fasting time is graphed in Fig. 1.
The body weight of the fasting group was significantly decreased as compared to
the control group. However, the fasting group gained weight rapidly after refeeding
started and became significantly heavier than the control group. Fig. 2 shows the
lifetime growth of both groups. Weight was taken just before fasting. The most
striking observation was that the average body weight of the fasting group was
increased as compared to that of the control group; body weights at 18, 20, 26, 28,
58 weeks were significantly different as indicated in Fig. 2. The fasting mice weighed
approximately 10% more than the control mice.

Fig. 1. Body weights of the
control, and 
 fasting groups before, during, and after the first fasting
period. The fasting period is shown in this graph as a striped square b. Values are mean (g) 9SD.
* P B0.05, ** PB 0.01, *** P B0.005 when compared with an age-matched ad libitum group.

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65

Fig. 2. The life-time growth curves in female B/W mice of the
control, and 
 fasting groups. Weights
were recorded at 2-week intervals. Body weights of each group were significantly different at 18, 20, 26,
28 and 58 weeks of age. * PB 0.05 when compared with an age-matched ad libitum fed group. The
growth curves of control and fasting groups terminated at 65 weeks, and 89 weeks, respectively, with the
death of the last mouse. The mice of the fasting group weighed approximately 10% more than those of
the control group. Results are shown as mean (g) 9SD.
Table 1
Influence of fasting on various organs in female (NZB×NZW) F1 mice at 26 Weeks of agea
Group

Body weight

Spleen
(×10−2)

Liver

Thymus
(×10−2)

Kidney
(×10−1)

Adrenal gland
(×10−3)

Control
Fasting

32.2 92.6
32.0 91.5

9.1 9 2.4
8.19 1.3

1.5 90.2
1.6 90.1

4.8 90.6
5.7 90.6*

1.1 9 0.1
1.4 90.1**

8.1 90.4
8.8 90.2**

a
Short-term repeated fasting effects on body weights and various organs in female B/W F1 mice
sacrificed at 26 weeks of age. Five animals were used for each group in this experiment. Each value
represents the mean (g) 9 SD.
* PB0.05.
** PB0.01, significantly higher than the corresponding control value.

3.2. Organ weights
At 26 weeks of age when both of the groups were on feeding phase, the body
weights of the representative mice, which are different mice from the survival
group, from each group showed no differences, however, the kidney, thymus and
adrenal gland weights of the fasting mice were significantly higher than those of the
control mice, as shown in Table 1.

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3.3. Proteinuria
Animals of the two groups were also compared with respect to proteinuria. The
mice fed ad libitum began to develop severe proteinuria at 22 weeks of age, and all
had developed proteinuria by 50 weeks (Fig. 3). The fasting mice, however,
gradually developed proteinuria after 30 weeks, and 90% showed proteinuria by 78
weeks of age.

3.4. Mitogen responses
At 26 weeks of age, no significant differences in responses to PHA, ConA or LPS
were observed between mice in the fasting and control groups. Fig. 4 shows the
results of the influence of fasting on spleen cell response to PHA, Con A and LPS.

3.5. NK cell acti6ity
NK cell activity was not different between the fasting and the control group
(12.5 9 2.2% vs. 12.79 4.3%, E:T ratio= 100:1).

3.6. MLR
There was no significant difference in MLR between the fasting and control
group (stimulation index=4.7 9 0.5 and 3.99 1.1, respectively).

Fig. 3. Effect of fasting on the cumulative progression to high grade proteinuria of female B/W mice
over the total life span: , control goup; 
, fasting group. Proteinuria was assayed just before the
fasting period at 2-week intervals. Positive proteinuria was designated as ] 2+.

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67

Fig. 4. Comparison of ConA, PHA, and LPS between the control 
 and fasting 
 mice at 26 weeks
of age. Spleen cells were cultured for 64 h at 37°C and then [3H]thymidine was added for an additional
8 h of incubation. Resutls are mean9SD of 5 mice.

Fig. 5. Survival rate of female B/W mice:
control group (n =10), and 
 fasting group (n = 10). The
mean times of death ( 9 SD) for control and fasting mice were 47.9 99.4 and 64.0 915.3 weeks,
respectively.

3.7. Sur6i6al data
Fig. 5 summarizes in graphic form the cumulative mortalities. Although no
differences in mitogen response, MLR, or NK cell activity were observed at 26
weeks of age, the mice that repeatedly fasted survived significantly longer than
the full-fed mice (P B0.005, with use of the Kaplan–Meier’s method). The 50%
point of mortality for the control group is 46 weeks, while the 50% point of
mortality for the fasting group throughout life is 67 weeks. Mean survival times
(9 SD) for fasting and control mice were 64.09 15.3 and 47.99 9.4 weeks,
respectively.

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4. Discussion
The present results show that the female B/W mice that repeatedly fasted had an
increased length of the time before disease onset and had moderately prolonged
longevity. It has been reported that, in all cases and regardless of calorie source,
mice fed 60% of normal caloric intake lived from two to three times longer than
their paired full-fed mice (Kubo et al., 1987). The effect of short term repeated
fasting on longevity in rodents is not so noticeable as the effect of ‘undernutrition
without malnutrition’; however, fasting manipulation clearly prolonged the life
span of autoimmune mice. Further, surprising to us was the finding that in spite of
the prolongation of life and a delay in the development of renal disease, the average
body weight of the fasting group was increased compared with that of ad libitum
fed mice, which probably indicates that the total caloric intake of the fasting mice
was higher than that of the controls. These findings make pressing the question of
how fasting manipulation influences mean and maximum life span and how it
increases the disease-free interval in these short-lived, autoimmune-prone mice.
Numerous hypotheses about the effects of caloric restriction have been proposed,
including improved immunological responsiveness (Weindruch and Walford, 1982;
Kubo et al., 1984a,b; Walford et al., 1987), the role of high plasma-free corticosterone concentration, the glycation hypothesis (Sabatino et al., 1991; Masoro et al.,
1992), the prevention of the age-associated decline in hsp 70 expression (Heydari et
al., 1993), the reduction of oxidative damage to macromolecules such as protein,
and DNA (Harman, 1956; Sohal, 1993; Sohal et al., 1994; Dubey et al., 1996; Yan
et al., 1997). However, few reports are found regarding short-term repeated fasting
manipulation. An intermittent feeding regimen (fed every-other-day), somewhat
similar to our fasting regimen, was reported by Goodrick and coworkers, however,
their studies were not so different from the concept ‘undernutrition without
malnutrition’, in that these animals showed decreased body weights (Goodrick et
al., 1982, 1983). We did not find any significant differences in immune function
between the fasting and control groups at 26 weeks of age, but the kidney, thymus,
and adrenal gland weights of the fasting mice were significantly higher than those
of the control mice, which implies that some immunoendocrine system changes
might have occurred during and after the short term repeated fasting. Some reports
have indicated that various immune-endocrine values changed during fasting. Wing
and coworkers showed that mice fasting for 48 or 72 h had increased resistance to
the intracellular pathogen Listeria monocytogenes (Wing and Young, 1980). They
suggested that the increased resistance resulted from enhanced activity of the
monocyte – macrophage cell line. We previously investigated the effects of acute
starvation on the immune system function of mice, and found that immune
function, including phagocytic activity of macrophages and T cell mitogen, was
enhanced by a short-period of starvation, but was suppressed by a long-period of
starvation (Kubo et al., 1982). The fasting regimen in the present experiment
probably has a tendency to enhance the immune function of the mice, but further
studies are needed to resolve the optimal fasting period necessary to achieve the
maximum possible life-span in the mice. Ehrenfried and coworkers found that an

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69

acute 48-h fast resulted in a marked induction of hsp70 mRNA levels in the
stomach of adult rats; these levels rapidly returned to near-baseline levels after
refeeding (Ehrenfried et al., 1996). They speculate that hsp70 mRNA elevation in
the gut may lead to subsequent increases of Hsp protein, which plays an important
cytoprotective role in the gut after an injury or stress. There are few clinical reports
with regard to starvation and fasting therapy. Murray and his colleagues who
studied famine victims, reported that the nomad populations had a low incidence of
clinically significant tuberculosis, malaria, and brucellosis during periods of starvation, however, the incidence of disease caused by these intracellular pathogens
increased dramatically after refeeding (Murray et al., 1975, 1976). Fasting therapy
was used mainly for the treatment of obese patients in USA; Wing and colleagues
who studied various immune parameters in obese subjects before and after fasting,
showed that blood monocyte bactericidal activity and NK cell activity were
enhanced by fasting (Wing et al., 1983b). On the other hand, it has also been an
effective treatment for psychosomatic patients in Japan (Yamamoto et al., 1979).
We previously investigated changes in the immunoendocrine system of patients with
psychosomatic disorders during fasting therapy (Komaki et al., 1997). Although the
total number of lymphocytes decreased during fasting, NK cell activity increased
significantly. Plasma cortisol and DHEAS concentrations also increased significantly. The percentage of CD4 cells was negatively correlated with cortisol concentrations during fasting. These findings indicate that fasting affects immune variables
such as T cell subsets and NK cell activity, at least in part through changes in
adrenal gland-related hormones. These experimental and clinical data indicate that
some endocrine – immune – neural changes occurred during the fasting time. Although the precise mechanism is not clear, these endocrine-immune-neural changes
may enhance certain functions of the mice defense system and contribute to
prolongation of the life span of B/W mice in spite of increased body weight.
Extensive further experimental analyses including CD4/CD8 (Fernandes et al.,
1997), Th1/Th2 balance (Iwakabe et al., 1998), various cytokines (Ryffel et al.,
1994; Chandrasekar et al., 1995; Fernandes et al., 1997; Spaulding et al., 1997),
oxidative molecular damage, hsp70, corticosterone, apoptosis (Holt et al., 1998)
and leptin (Ahima et al., 1996), as well as behavioral function (Dubey et al., 1996)
and cancer incidence (Weindruch et al., 1992) will be required to clarify the role of
fasting.
In conclusion, the present report demonstrates that the effects of short term
repeated fasting prolongs life spans and inhibits the development of renal disease in
short-lived, autoimmunity-susceptible B/W mice in spite of heavier body weight
than found in ad libitum fed mice.

Acknowledgements
This study was partially supported by grants from Smoking Research
Foundation.

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