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1
Comparison of Intermittent Fasting versus Caloric Restriction in Obese
Subjects: A Two Year Follow-up
Fehime Benli Aksungar, Mustafa Sarıkaya, Abdurrahman Coskun, Mustafa Serteser, Ibrahim
Unsal
Running Title: IF vs CR in obese subjects: A two year follow-up
Fehime Benli Aksungar, MD, Assoc. Prof.
Acıbadem University, School Of Medicine, Department of Biochemistry, Istanbul, Turkey
Acıbadem Labmed Clinical Laboratories, Istanbul, Turkey
Mustafa Sarıkaya, MD, Assist. Prof.
Maltepe University, School of Medicine, Department of Physiology, Istanbul Turkey
Abdurrahman Coskun, MD, Prof.
Acıbadem University, School Of Medicine, Department of Biochemistry, Istanbul, Turkey
Acıbadem Labmed Clinical Laboratories, Istanbul, Turkey
Mustafa Serteser, MD, Prof.
Acıbadem University, School Of Medicine, Department of Biochemistry, Istanbul, Turkey
Acıbadem Labmed Clinical Laboratories, Istanbul, Turkey
Ibrahim Unsal, MD, Prof.
Acıbadem University, School Of Medicine, Department of Biochemistry, Istanbul, Turkey
Acıbadem Labmed Clinical Laboratories, Istanbul, Turkey
Corresponding Author:
Fehime Benli Aksungar, MD, Assoc. Prof.
Acıbadem Labmed Clinical Laboratories,
Acıbadem University, İçerenköy Kayışdağı Caddesi
34752 Ataşehir, Istanbul, Turkey
e-mail: fehime.aksungar@acibadem.edu.tr
Tel: +90 532 172 7854
2
Conflict Of Interest: None
Acknowledgements: We want to thank to our subjects who made this work come to life with
their adherence to our programme for two years.
3
Abstract
Objectives: Caloric restriction (CR) is proven to be effective in increasing life span and it is well
known that, nutritional habits, sleeping pattern and meal frequency have profound effects on
human health. In Ramadan some Muslims fast during the day-light hours for a month, providing
us a unique model of intermittent fasting (IF) in humans. In the present study, we have
investigated the effects of IF versus CR on the same non-diabetic obese subjects who were
followed for two years according to the growth hormone (GH)/Insulin like growth factor (IGF)-1
axis and insulin resistance
Design: Single-arm Interventional Human Study
Participants: 23 female subjects (Body Mass Index (BMI) 29-39, aged between 28-42years)
Setting: Follow-up is designed as 12 months of CR, after which there was a month of IF and 11
months of CR again, to be totally 24 months. Subjects’ daily diets were aligned as low calorie
diet during CR and during the IF period, the same subjects fasted for 15 hours in a day for a
month and there was no daily calorie restriction. Nutritional pattern was changed as 1 meal in the
evening and a late supper before sleeping and no eating and drinking during the day light hours
in the IF model. Subjects made brisk walking twice a day during the whole follow-up including
both CR and IF periods. BMI, Blood glucose, insulin, TSH, GH, HbA1c, IGF-1, Homa-IR and
urinary acetoacetate levels were monitored once in three months and twice in the fasting month.
Measurements and Results: While subjects lost 1250 ± 372g monthly during the CR, in the IF
period, weight loss was decreased to 473 ±146 g. BMI of all subjects decreased gradually and as
the BMI decreased, glucose, HbA1c, insulin, Homa-IR and TSH levels were decreased. GH
levels were at baseline at the beginning, increased in the first six months and stayed steady
during the CR and IF period than began decreasing after the IF period, while IGF-I increased
gradually during the CR period and beginning with the 7th day of IF period, it decreased and kept
on decreasing till the end of the follow-up. Urinary acetoacetate levels were higher during the IF
period suggesting a constant lipid catabolism.
Conclusion: Our results suggest that, CR affects metabolic parameters positively which will
help especially pre-diabetic and insulin resistant patients without any pharmacological approach.
In addition IF without calorie restriction can enhance health and cellular resistance to disease
without losing weight and those effects may be attributed to different signalling pathways and
circulating ketones during IF. Changes observed during IF are probably due to the changes in
eating and sleeping pattern and thus changes in metabolic rhythm.
4
Keywords: Fasting, Intermittent fasting, Calorie restriction, Low carbohydrate, Insulin
resistance
Introduction:
High caloric intake and a sedentary life style are well known environmental risk factors having
an impact on life-span. Caloric restriction (CR) is proven to be effective in increasing life-span
and disease resistance in rats, mice, and Rhesus monkeys in recent studies (1-3). Caloric
restriction models generally refer to a 30-40% decrease in daily caloric intake (3,4). On the other
hand, intermittent fasting (IF) is also shown to have positive impact on life span (4). IF model in
mice is defined as every other day feeding without caloric restriction (4). Although there is no
consensus and defined IF model in humans, a prolonged intermittent fasting time for a month
during Ramadan provides a good IF model. In Ramadan every year, depending on the time of
year, Muslims fast for approximately 15 hours during the day and eat at night without caloric
restriction for a month. Many physiological changes are observed during Ramadan fasting that
are likely due to the changes in eating and sleeping patterns. Many societies have recognized the
beneficial effects on health and longevity of limited food intake for certain periods of time, either
for religious reasons or when the food supply was insufficient (5). In the present study, we have
investigated the effects of IF versus CR (40%) on obese non-diabetic subjects regarding the
GH/IGF axis, insulin resistance.
Methods:
Twenty-three female subjects without diabetes whose body mass indices (BMI) were 29-37
(mean ± SD, 34.15 ± 2.15 and aged 28-42 (mean ± SD, 36 ± 3.12) years were followed for 24
months. The study design and plan were thoroughly explained to the patients orally and in
writing, and written informed consent was obtained from all patients and the study was approved
by the ethics committee of Maltepe University, School of Medicine. The study has begun with
30 patients, 7 of whom, were excluded since they were not adherent to the diet regimens.
Follow-up is designed as 12 months of CR, after which there was a month of IF (Ramadan) and
11 months of CR again, to be totally 24 months. Patients’ routine daily diets were followed for a
month with a questionnaire and daily caloric intake was calculated (mean ± SD, 2610 ± 409
calories) before the study began. Mean caloric intake of all the subjects was aligned to be 40%
less during the study, which was followed and recorded by weekly interviews. The main
5
restriction was from carbohydrates (CH); daily CH intake was restricted to 120g and total caloric
intake was aligned to be nearly (mean ± SD) 1518 ± 212 calories during the study. Subjects
made brisk walking twice a day (am and pm) for an hour in total. This was the CR model. The
same subjects fasted for 15 hours/day for 30 days during the month of Ramadan. This model
was used as the IF model, which is also called “Prolonged intermittent fasting”. During IF, there
was no daily calorie restriction. The nutritional pattern was changed to one meal in the evening
and a late supper one hour before sleeping, with no eating or drinking during the day. Daily brisk
walking sessions were continued and subjects continued their daily activities during the fasting
days.
The follow-up began 12 months before the fasting month in 2014 and ended at June 2015. Nine
fasting blood and urine samples were taken during the follow-up (once in three months before
Ramadan, at the end of the first week (7th day), and the last week (21st day) of Ramadan Month
and three more samplings after Ramadan), sampling design and frequency are summarized in
table 1. Totally 9 samplings were performed. Glucose, insulin, thyroid stimulating hormone
(TSH), growth hormone (GH), hemoglobin (Hb)A1c, insulin like growth factor (IGF)-1, Homa-
IR, and urinary acetoacetate levels were monitored. Twenty-four hour urine samples were
collected for six times during the follow-up and 24 hour urinary ketones were determined semi-
quantitatively. Additionally, 24 hour urinary volumes were monitored in order to determine any
kind of dehydration during IF. Glucose was analyzed by hexokinase/G6PDH method in RxLMax
(Siemens/Germany) autoanalyzer. Insulin, TSH, GH were measured by
electrochemiluminescence immunoassay (ECLIA) method in Roche/ Cobas (Germany) systems,
and the analytic sensitivity of GH reagent was 0.002 ng/mL. Homa-IR was calculated as
multiplying fasting serum insulin (FPI) by fasting serum glucose (FPG), then dividing by the
constant 22.5, i.e. HOMA-IR =(FPI×FPG)/22.5 (6), IGF-1 was measured by chemiluminescence
immunoassay method (CLIA) in IDS-ISYS (Immunodiagnostic Systems-France) and finally
HbA1c was measured by HPLC method (BioRad-Variant-England/USA) and only five
samplings were made for HbA1c during the entire follow-up (Table 2). All blood samples were
taken after 12 hour fasting, and during Ramadan, great attention was paid to wait for 12 hours
after the last meal.
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Statistical analysis was performed by using repeated-measures ANOVA since each subject
served as her own control. Post hoc testing was performed with Dunn’s multiple comparison test
and p values <0.05 were considered to be statistically significant.
Results:
While subjects lost 1250 ± 372g on average in a month during the CR follow-up, in the IF
period, weight loss decreased to 473 ± 146g. Twenty-four hour urinary volumes did not change
significantly when the IF period was compared to CR period (data not shown).
As the BMI decreased, glucose, insulin, and Homa-IR levels decreased significantly with CR.
During the IF period while glucose levels continued to decrease, insulin levels remained at the
same level (Fig 1 and 2). TSH levels were 3.15 ± 0.85 uIU/mL on average at the beginning, and
decreased gradually with decreasing BMI in all patients (Table 2). HbA1c percentage begun the
study with 6.46 ± 1.26% on average and after 24 months it was 4.85 ± 1.20 % (Table 2). GH
levels were at baseline at the beginning, increased in the first six months, stayed steady during
the follow-up and IF period then decreased after the IF period (Fig 3), while IGF-I increased
gradually during the follow-up and beginning at the 7th day of IF period, it decreased and kept
on decreasing till the end of the follow-up (Fig 3). Urinary acetoacetate levels were monitored
six times, which were higher during the IF period, suggesting an increased lipid catabolism
during the fasting days (Table 2).
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Table 1
Sampling Scheme and Applied Study
Table 2
Changes in Metabolic Parameters During The Follow-up
Sampling #
1
2
3
4
5
6
7
8
9
BMI (kg/m2)
34.15 ± 2.12
32.47 ± 3.10*
31.15 ± 3.31*
29.62 ± 2.01
29.32 ± 2.19
28.04 ± 4.50
27.81 ± 3.16*
25.13 ± 2.18*
26.02 ± 1.14
HbA1c (%)
6.46 ± 1.26
6.11 ± 1.91
5.71 ± 2.54*
4.93 ± 1.83*
4.85 ± 1.20
HOMA-IR
(Calculated)
12.92 ± 2.51
10.03 ± 5.10*
7.97 ± 2.19*
5.61 ± 3.09*
5.48 ± 2.81
4.87 ± 2.18*
4.33 ± 1.19
4.41 ± 1.04
4.26 ± 1.31
TSH (µIU/mL)
3.15 ± 0.85
3.01 ± 1.13
2.83 ± 1.82*
2.56 ± 1.23
2.50 ± 1.05
2.56 ± 1.92
2.16 ± 1.15*
2.01 ± 1.84
2.24 ± 1.16
Acetoacetate
(mg/24 h urine)
137.4 ± 27.5
146.6 ± 24.0
373 ± 35.08*
284.91 ± 35.27*
163.62 ± 22.10*
153 ± 47.25*
Sampling #, 1; month 0, 2; month 4, 3;month 7, 4;month 10, 5; 7th day of Ramadan month, 6; 21st day of
Ramadan month, 7; month 17, 8;month 20, 9; month 24. HbA1c sampled for 5 times and urinary acetoacetate
sampled for 6 times during the follow-up. Values indicate mean ± SD. *Statistically different (p<0.05), when
compared to previous sampling. Normal fasting reference ranges of analytes are as follows, HOMA-IR <2.7, TSH
0.24-4.0 µIU/mL, Acetoacetate <25mg/day
Date of Sampling
Study Model
Number of Sampling
July 2013 (Month 0)
N/A
#1
October 2013 (Month 4)
CR
#2
January 2014 (Month 7)
CR
#3
April 2014 (Month 10)
CR
#4
Agust 2014 (7th Day of
Ramadan Month)-Month 14
IF
#5
Agust 2014 (21st Day of
Ramadan Month)-Month 14
IF
#6
November 2014 (Month 17)
CR
#7
February 2015 (Month 20)
CR
#8
June 2015 (Month 24)
CR
#9
The first sampling was performed as the basal values of the subjects at month 0,
there was no intervention at that month (N/A). CR, Caloric restriction model; IF,
Intermittent fasting model.
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Figure 1
Figure represents the changes in glucose levels during the follow up, with the changes in BMI. Sampling number, 1;
month 0, 2; month 4, 3;month 7, 4;month 10, 5; 7th day of Ramadan month, 6; 21st day of Ramadan month, 7;
month 17, 8;month 20, 9; month 24. Values indicate mean ± SD. *Statistically different (p<0.05), when compared
to previous sampling. Normal fasting reference ranges of glucose is 70-99 mg/dL. BMI body mass index.
123 115
102 97 95
87
92 89 90
34,15
32,47
31,15
29,62 29,32 28,04 27,81
25,13 26,02
0
5
10
15
20
25
30
35
-10
10
30
50
70
90
110
130
150
123456789
BMI (kg/m2)
Glucose (mg/dL)
Sampling Number
*
*
*
*
*
*
*
9
Figure 2
Figure represents the changes in insulin levels during the follow up, with the changes in BMI. Sampling number, 1;
month 0, 2; month 4, 3;month 7, 4;month 10, 5; 7th day of Ramadan month, 6; 21st day of Ramadan month, 7;
month 17, 8;month 20, 9; month 24. Values indicate mean ± SD. *Statistically different (p<0.05), when compared
to previous sampling. Normal fasting reference ranges of Insulin 3-25 µIU/mL, BMI, body mass index.
36,01
30,56 28,71
26,14 25,12 25,27 23,61
20,18 20,41
34,15
32,47 31,15 29,62 29,32 28,04 27,81
25,13 26,02
0
5
10
15
20
25
30
35
40
0
5
10
15
20
25
30
35
40
45
50
1 2 3 4 5 6 7 8 9
BMI (kg/m2)
Insulin (µU/mL)
Sampling number
*
*
*
*
*
*
*
10
Figure 3
Growth hormone (GH-right y-axis) and IGF-1 (left y-axis) changes during the follow-up (values show the mean
concentrations of all the subjects for the sampling period). Numbers on the x-axis indicates the sampling number;
Sampling #, 1; month 0, 2; month 4, 3;month 7, 4;month 10, 5; 7th day of Ramadan month, 6; 21st day of
Ramadan month, 7; month 17, 8;month 20, 9; month 23. *Statistically different (p<0.05), when compared to
previous sampling. GH (Growth Hormone) reference ranges are 0-4 ng/mL. IGF-1 (Insulin-like growth Hormone)
reference ranges are 110-453 ng/mL
Discussion:
There are great struggles to extend healthy lifespan in humans. Longevity-extending studies have
focused on GH, IGF-1, and insulin axis, and their effects on survival and healthy aging. It has
been thoroughly demonstrated that nutritional habits, sleeping patterns, and meal frequency have
profound effects on human health (7,8). Caloric restriction and IF are both shown to be potential
diets for healthy body and brain aging (5). A reduction in food intake without malnutrition, as in
the CR model, may decrease the activity of nutrient-signaling pathways that mimic the periods of
food shortage in nature (1), which in turn may activate cellular stress-resistance pathways.
Furthermore, in a study by Anson et al., it is shown that without caloric restriction, intermittent
118
194
238 242 227 216
182
157 142
0,502
0,791
0,919 1,012 1,023 1,031 0,962
0,615
0,472
0
0,25
0,5
0,75
1
1,25
1,5
1,75
2
2,25
2,5
0
50
100
150
200
250
300
350
400
1 2 3 4 5 6 7 8 9
GH (ng/mL)
IGF-I (ng/mL)
Sampling Number
*
*
*
*
*
*
*
*
*
*
11
fasting (IF) had beneficial effects on neuronal resistance and aging, showing the same effects as
the CR model without a significant weight loss in mice (4).
In the present study, after the 24 month follow up, BMI, fasting glucose, insulin, HbA1C, and
HOMA-IR levels of all subjects decreased gradually overtime, suggesting long term CR and
physical exercise have great beneficial effects on the health of obese patients. Decreasing
HOMA-IR and insulin levels together with decreasing HbA1C concentrations show that the risk
of type 2 diabetes and metabolic syndrome is significantly reduced by long term CR. This
beneficial effect has been well reported by previous studies and already applied to therapy (9,
10). The unknown is, whether prolonged IF has similar influences on the health and risk of
metabolic diseases. To date, there have been limited comparison studies on humans to
understand the effects of long term CR and IF. The present study showed both long term caloric
restriction and a short term IF model have beneficial effects on human health while they have
different influences on the IGF-GH-insulin axis. During CR, IGF-1 levels increased gradually,
while during IF, with the 7th day of fasting period there was a decline which continued till the
end of the follow-up (Fig. 3). Although IF lasted only for one month and again a CR period
began, its effects seem to continue for at least 11 months till the end of the study. This decline
was not clear in GH levels, it began to decrease after the IF period. These findings suggest that
there may be a difference in the signaling pathways of IF and CR according to the GH-IGF-1and
insulin axis.
GH secretion is regulated by a complex network of neurotransmitters and neuropeptides. It is
mainly under control of GH-releasing hormone and somatostatin besides other hormones,
including sex steroids, glucocorticoids, gastrointestinal hormones and metabolic factors.
Nutrition and metabolism play a key role in the regulation the GH-IGF-1 axis. It is shown that in
obesity, stimulated GH peak is negatively correlated with BMI (11).
IGF-1 mediates many of the somatic effects of GH and GH concentrations are controlled by a
negative feedback due to free IGF-1 levels. Recent data have shown that IGF-1 has insulin-like
effects on the peripheral uptake of glucose and fatty acids (12). Most cells that express the IGF-1
receptor (IGF-IR) and IGF-1 in circulation are mostly produced by the liver, and its levels
largely regulated by GH. However, other factors may also affect hepatic IGF-1 synthesis,
including nutritional status, such as caloric intake and protein consumption, insulin, and
12
inflammatory cytokines (13-15). There is growing evidence that shows increased IGF-1 levels
have been associated with reduced insulin resistance (16). In the present study increase in IGF-I
levels and the decrease in HOMA-IR values during CR are consistent with these findings.
Administration of recombinant IGF-1 was found to reduce serum glucose levels and improve
insulin sensitivity in healthy adults as well as in insulin resistant subjects and type 2 diabetics
(17). This insulin sensitizing effect of IGF-1 may not only be due to its GH suppressing effect,
but also due to independent IGF-1 effects. It has been reported that insulin like growth factor
binding protein (IGFBP)-3, the most abundant IGFBP in circulation (among the known six of
them), may play a role in glucose homeostasis by binding to a nuclear receptor that interacts with
peroxisome proliferator activated receptor-gamma (PPAR-), a nuclear protein involved in the
regulation of glucose and lipid metabolism (18,19). The metabolic effects of IGFBP-3 are largely
opposite of those of IGF-1 (20). Transgenic animal data demonstrate that over expression of
IGFBP-3 is associated with fasting hyperglycemia and impaired glucose tolerance in mice (21).
In the current study, the increase in IGF-1 during CR may result in decreased levels of IGFBP-3
due to binding, resulting in increased insulin sensitivity. We have shown in this particular study
that GH and IGF-I concentrations were down regulated during and after IF period. GH/IGF-I
signaling with nutrition has different aspects. A decreased GH/IGF-I signaling is a common
characteristic of aging where as a constitutively decreased pathway extends longevity (2).
GH/IGF-I pathway down modulation may reflect a defensive response for minimizing cell
growth and metabolism during systemic damage trying to escape from death. It is shown that
organisms with a constitutively decreased GH/IGF-I pathway can survive longer (2). This
GH/IGF-I axis down regulation during and after the IF period may be due to low energy intake
and adaptation of the metabolism.
During IF, BMI did not change significantly, but after the IF period, it again began to drop with
ongoing CR, suggesting day-long fasting model (IF) cannot be assumed as a weight loss
program, but can be thought of as a detoxification/regeneration or a longevity-extending process
since IGF-1 levels were lower during IF and also circulating ketones were elevated which stayed
high during nine months after IF period. During IF, increased reliance on β-oxidation of fatty
acids for meeting energy needs as a response to reduced availability of nutrients, may be
associated with improved metabolic homeostasis. A shift from carbohydrate to fatty acid
utilization in response to reduced nutrients is supposed to be an important metabolic adaptation
13
of mitochondrial function and one of the key mechanisms of extended longevity (22). Mice on
an IF regimen have been shown to develop a two-fold increase in the fasting serum
concentrations of ketones compared with mice on CR (5). This shift to ketogenesis may play a
direct role in the cytoprotective effects of IF, since it has been reported that rats that were fed a
ketogenic diet exhibited increased resistance to seizures and β-hydroxybutirate itself can protect
neurons in models of Alzheimer’s and Parkinson’s disease (23,24). Changes in the IF period
may be due to omitting at least two meals when the body is metabolically active. Moreover,
glycogen stores of all cells must be regenerated every day since there was absolute fasting during
the day for 15 hours. Regarding IGF-1, recent data have shown an inverse correlation between
levels of IGF-1 with CRP and other cytokines, indicating IGF-1 may be involved in the altering
inflammatory response to insulin resistance and its progression to type-2 diabetes (25).
Furthermore, it is shown that circulating levels of adiponectin, an important anti-inflammatory
adipokine, are elevated in long-lived, calorie restricted mice, whereas the expression of
proinflammatory cytokines, IL-6, and TNF-α are reduced (26). In the current study, although
concentration of serum adiponectin was not determined in the subjects, one of the previous
studies of the researchers indicated that IL-6 and CRP levels significantly decreased in an IF
model (7). Adiponectin, in addition to its anti-inflammatory properties, also promotes B-
oxidation of fatty acids and enhances insulin sensitivity by activating adenosine monophosphate-
activated protein kinase (27). Moreover, cellular stress-resistance pathways may be activated by
IF; It has been shown that levels of chaperones and neurotropic factors increased in rats
maintained on an IF regimen (4). In our IF model, during the fasting day light hours, catabolism
is active and on gorging, anabolism is active. Alternating periods of anabolism and catabolism
may play a mechanistic role in triggering cellular stress-resistance pathways and repair of
damaged cells. Hence the beneficial effects of this IF model may be due to the stress associated
with fasting rather than the caloric intake. Since it is shown that IGF-1 signaling is
neuroprotective (28,29), it is important to determine the mechanisms by which CR and IF
differentially affect IGF-1 levels and insulin signaling and their influence on energy metabolism,
disease resistance, and longevity. Therefore, it must be well investigated in future studies
whether a reduction in calorie intake is the only dietary method by which to increase healthy
longevity or whether IF without calorie restriction might have beneficial effects similar to CR.
14
This study had certain limitations one of which is we did not have a different control group, we
thought it is important to see the changes particularly on the same subjects, since they served as
their own controls and by this way we had avoided the inter-individual variation in the monitored
parameters. Second limitation is about the sampling; We had no sampling just before the IF
period, the last sampling was three months ago. Since CR had began 12 months ago before the IF
period, which means subjects had already reached the basal levels of all metabolic parameters,
we assessed the results comparing to these measurements. Finally the last limitation is about the
sample size of the study which seems to be small, we had begun the study with 30 subjects but as
stated before 7 of them had to be excluded because of the lack of adherence to the diet regimen
since it is a really challenging task for the individuals for 24 months. But still 23 subjects seem to
be proper enough for reflecting the differences between CR and IF model. There is limited data
on humans in IF model and in our opinion the present study adds valuable data to this area.
In conclusion, this study demonstrated, constantly losing weight with CR and physical exercise
affects metabolic parameters positively, which will particularly aid pre-diabetic and insulin
resistant patients leading to non-pharmacologic approaches. Moreover one of the most striking
result of the study is that IF can enhance health and cellular resistance to disease by different
mechanisms, even if the fasting period is followed by a period of overeating such that overall
caloric intake is not decreased. Further human studies on a molecular basis are needed in order to
reveal the signaling mechanisms that differentially occur during intermittent fasting and CR to
support these results.
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