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Does Intermittent Fasting Improve Microvascular Endothelial Function in
Healthy Middle-aged Subjects?
Fatemeh Esmaeilzadeh* and Philippe van de Borne
Department of Cardiology, Erasme Hospital, Universite Libre de Bruxelles (ULB), 808 Lennik Street, 1070 Brussels, Belgium
*Corresponding author: Fatemeh Esmaeilzadeh, MD, Department of Cardiology, Erasme Hospital, Universite Libre de Bruxelles (ULB), 808 Lennik Street, 1070
Brussels, Belgium, Tel: +32 (0)2 555 3907; Fax: +32 (0)2 555 6652; E-mail: f.esmaeilzadeh@erasme.ulb.ac.be
Received date: August 13, 2016; Accepted date: September 7, 2016; Published date: September 14, 2016
Copyright: © 2016 Esmaeilzadeh F, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited.
Abstract
Background: Reduced endothelial nitric oxide bioavailability, a hallmark of endothelial dysfunction, is commonly
encountered in cardiovascular diseases. Intermittent fasting reduces serum markers of oxidative stress, while nitric
oxide levels may rise. Whether this translates into persistent improvements in endothelial function is unknown. The
aim of the study was to address the effects of intermittent «Ramadan-type» fasting on endothelial function, nitric
oxide bioavailability, biological parameters and blood pressure.
Methods: We tested this hypothesis in fourteen healthy middle-aged male subjects, using a prospective case-
controlled study design. Microvascular endothelial function of skin vessels was evaluated with a laser Doppler
imager, Before-fasting, after thirty days of fasting, and one month thereafter (Post-fasting). Endothelial dependent
and independent dilatations were assessed by acetylcholine and sodium nitroprusside iontophoresis, respectively.
The hyperemic response to heating after a specific nitric oxide-synthase inhibitor L-N-arginine-methyl-ester
administration, versus a saline solution, allowed further characterization of nitric oxide-mediated vasodilation. Blood
pressure, body mass index, metabolic parameters were determined in all subjects.
Results: Blood pressure decreased, while blood glucose and LDL-cholesterol increased during fasting (all p<0.05
vs. Before-fasting). Body mass index did not change. Hyperemic skin reactions assessed by acetylcholine increased
during Fasting and Post-fasting, while sodium nitroprusside-induced hyperemia and nitric oxide-related vasodilation
in response to heating increased during Fasting only (all p<0.05 vs. Before-fasting). Rises in serum triglycerides and
blood urea nitrogen during fasting blunted nitric oxide-related vasodilation improvement upon heating (r=-0.55 and
-0.60 respectively, p<0.05). These parameters did not change over time in thirteen matched controls.
Conclusion: Intermittent fasting improved endothelial and non-endothelial dependent vasodilations and decreased
blood pressure. Increased nitric oxide bioavailability during this period was negatively related to rises in serum
triglycerides and blood urea nitrogen.
Keywords: Intermittent fasting; Microvascular endothelial function;
Nitric oxide; Laser Doppler owmetry; Ramadan fasting; Healthy
middle-aged men
List of Abbreviations:
NF-kB: Nuclear factor kappa-light-chain-enhancer of activated B
cells; BMI: Body Mass Index, NO: Nitric Oxide; Ach: Acetylcholine;
SNP: Sodium Nitroprusside; LDI: Laser Doppler Imager; SkBF: Skin
Blood Flow; BSL: Baseline; μA: Microamperes; L-NAME: L-N-
arginine-methyl-ester; NaCl: Normal saline; TG: Triglycerides; Chol:
Total Cholesterol; HDL: High-density Lipoprotein; LDL: Low-density
Lipoprotein; BUN: Blood Urea Nitrogen; Cr: Creatinine; Tot. Bil: Total
Bilirubin; PU: Perfusion Unit; AUC: Area under the curve; NS: Not
Signicant; BP: Blood Pressure; Sp: Saline; EDHF: Endothelium-
Derived Hyperpolarizing Factor
Introduction
e vascular endothelium has a crucial role in the regulation of
normal blood ow and platelet activity [1]. Increased oxidative stress is
linked with endothelial dysfunction in atherosclerosis, and may have
an important role in the pathogenesis of cardiovascular events [2].
ere is growing evidence that fasting reduces oxidative damage and
inammation [3-7]. e exact mechanism responsible for this
observation is however not known [6]. Reduced energy intake, which
lessens oxidative stress formation in the mitochondria, and, as a result,
reduces oxidative damage to the cells, may play a role [6-8]. e most
striking evidence of the anti-inammatory eect of fasting relies on at
least four controlled studies in patients with rheumatoid arthritis, two
of which were randomized [9-11]. is was also observed in asthma
patients [12]. Experimental studies in mice maintained on intermittent
fasting diet showed increased resistance to oxidative insults [13]. In
rats, alternate-day fasting also protected their hearts against
inammation and brosis by inhibiting oxidative damage and NF-kB
(nuclear factor kappa-light-chain-enhancer of activated B cells)
activation [14]. ese observations could explain why caloric
restriction extends lifespan and delays the onset of age-related diseases
in a wide spectrum of organisms [15]. ese results are in contrast to
the eects of recurrent religious fasting on human health, where
studies provided either such heterogeneous results that no overall
conclusion could be reached [16], while others reported improvements
in body mass index (BMI), lipid prole, and blood pressure [17-20].
Biology and Medicine
Esmaeilzadeh and van de Borne, Biol Med
(Aligarh) 2016, 8:6
DOI: 10.4172/0974-8369.1000337
Research Article OMICS International
Biol Med (Aligarh), an open access journal
ISSN: 0974-8369
Volume 8 • Issue 6 • 1000337
Reasons for these discrepancies are likely the lack of standardization of
the meals taken from sunrise to sunset, the dierent moments of the
year where fasting take place, and the variable durations of fasting,
depending of the latitudinal distance from the equator. Uncontrolled
studies have nevertheless been able to show that intermittent fasting
decreased oxidative stress [4,5,21]. Lessened reactive oxidative species
could plausibly cause a rise in nitric oxide (NO) levels and thereby
improve endothelial function [22]. is was suggested by a previous
uncontrolled study, where recurrent intermittent fasting for more than
25 days in cardiovascular disease patients increased serum NO, while
serum asymmetric dimethylarginine, the naturally occurring
endogenous inhibitor of NO synthase, decreased [23].
Whether fasting can result in functional improvements of
microvasculature is not known. Using the previously cited studies as a
background, we decided to test the hypothesis that recurrent
intermittent «Ramadan-type» fasting improves endothelial
microvascular function in healthy subjects. Endothelial function was
evaluated Before-fasting, aer 1 month of Fasting and 1 month aer
fasting cessation (Post-fasting). Endothelium dependent and
independent vasodilation were assessed by iontophoresis of
acetylcholine (Ach) and sodium nitroprusside (SNP), respectively
[24,25], while vasodilatation in response to local heating (in the
presence or absence of a NO synthase inhibitor) determined the
contribution of NO to skin microvascular endothelial function [26].
ese repeated measurements were also obtained in a matched non-
fasting control group. We are not aware of a previous prospective
controlled study on intermittent fasting and endothelial function.
Materials and Methods
Subjects
Assuming a level of signicance at 5% and a study power at 80%,
and according to previous validation and interventional studies in our
laboratory [26,27], it was estimated that 15 subjects had to participate
to demonstrate a threefold increase in the contribution of NO to the
microvascular response to heating with fasting. Based on their health
status, a total of 27 subjects were considered eligible to participate in
the study, among of 60 volunteers. We compared 14 healthy male
volunteers would intended to perform fasting during the Ramadan
period, to 13 subjects matched for gender, ethnicity, age and BMI. e
study protocol (reference: P2014/232; B406201421283, Registered June
16, 2014) was in agreement with the Ethics principles of Helsinki and
was approved by the Ethics committee of the Erasme University
Hospital. e study was conducted from June 20th to September 1st,
2014. e fasting regimen started on June 28th until July 27th, 2014. A
written informed consent was obtained from all subjects.
Study design
We designed a prospective case-controlled study to test our working
hypothesis. All subjects were older than 18 years, nonsmokers, had no
concomitant disease and took no medications. is prevented that
overnight smoking and medication intake aect our ndings. Because
the menstrual period may impede religious fasting and aect
endothelial function [28-30], only male subjects were allowed to
participate in the study. e fasting group underwent 19 hours of
intermittent fasting for 26 ± 0.5 consecutive days, and was asked to
consume no more than one large meal aer sunset and one lighter
meal before sunrise, in order to reduce caloric intake variability in our
study [31]. All subjects were asked to keep their usual lifestyle and
daily activities during the study. Experimental conditions were
controlled by enrolling the subjects with similar lifestyle and activity, in
order to minimize the impact of confounding variables.
Casual humeral blood pressure was determined by E.F. in the sitting
position, using a device (WelchAllyn, USA) with a cu size of 250-340
mm. Aer 5 minutes of rest, 2 measures on the non-dominant arm,
separated by 1 minute, were averaged. Anthropometric measurements
and fasting blood samples were obtained from all subjects on the day
of the microvascular ow assessment. ree measures were obtained in
the fasting group: before the beginning of the fasting regimen, aer 26
± 0.5 days of fasting, and 34.4 ± 2.5 days aer fasting termination (i.e.
these measurements are called respectively «Before-fasting», «Fasting»
and «Post-fasting» throughout the manuscript). Two measures,
separated by 28 ± 1.4 days, were performed in the control group. e
moment of the day where these measurements were performed was
kept identical in each subject and group throughout the study.
Microvascular blood ow was assessed by a laser Doppler imager
(LDI). All subjects abstained from meals for 10 ± 2 hours and from
alcohol and coee beverages for at least 24 hours prior to each LDI
session. ey were asked not to wash their forearms on the morning of
the experiment day and to avoid non-steroidal anti-inammatory
drugs for at least 3 days before each test. Two subjects could not
participate to the Post-fasting measurements, because of a knee
accident in one and vacation in the other one.
Microvascular endothelial function evaluation
All measurements were performed in a quiet room, in the supine
position under carefully standardized conditions. e subjects were
not allowed to sleep during the experiments. e ambient temperature
in the room achieved by the air conditioner was 23 ± 1°C.
Cutaneous microcirculatory blood ow was assessed by a LDI
(Moor Instruments, version 5.3d soware, Axminster, United
Kingdom) to measure the skin blood ow (SkBF) in a region of interest
corresponding to a surface of skin of 3.8 cm². e reproducibility and
accuracy of this method for endothelial function measurement has
already been tested previously in our laboratory [26]. e servicing
and calibration of the laser Doppler machine were made prior to
beginning the measurements. Before beginning the measurements, and
according to LDI guidelines, specic care was taken to create similar
experimental conditions to ensure regional and temporal
reproducibility [32]. Measures were performed at baseline (BSL) and
during hyperemic tests. For each measure, 12 scans were acquired,
where the 2 rst scans corresponded to the BSL cutaneous ow.
Twenty minutes before the measurement, 5% EMLA cream® (2.5%
lidocaine and 2.5% prilocaine; AstraZeneca, London, UK) was applied
to the skin surface in order to limit any non-specic vasodilation
induced by the electric current [33]. Firstly, we performed Ach and
SNP- induced hyperemia by administering these molecules
percutaneously using dedicated iontophoresis chambers (ION6; Moor
Instruments Ltd, Axminster, United Kingdom). Ach and SNP solutions
were prepared to obtain a nal concentration of 2 g/dL in deionized
water, and 2.5 ml of these solutions was introduced into the cathode
(Ach electrode) and the anode (SNP electrode) chambers. Electric
current was generated by an iontophoresis controller (MIC2, Moor
Instruments Ltd, Axminster, United Kingdom), which was set to apply
a current of 100 microamperes (μA) for 20 minutes. Ach and SNP
iontophoresis were continued for 26 minutes in order to obtain a
maximal skin vasodilation.
Citation: Esmaeilzadeh F, van de Borne P (2016) Does Intermittent Fasting Improve Microvascular Endothelial Function in Healthy Middle-aged
Subjects?. Biol Med (Aligarh) 8: 337. doi:10.4172/0974-8369.1000337
Page 2 of 9
Biol Med (Aligarh), an open access journal
ISSN: 0974-8369
Volume 8 • Issue 6 • 1000337
We also assessed skin hyperemia response to local heating according
to our previously described methodology [26]. In summary, aer a
skin pre-treatment in 2 adjacent skin areas either by L-NAME (L-N-
arginine-methyl-ester, 20 mmol/L) or NaCl (Normal saline 0.9 g/dL,
Baxter®) iontophoresis, the skin was heated to 44°C using dedicated
skin heater electrodes and a temperature monitor (SH02, Moor
Instruments Ltd., Axminster, United Kingdom). Heating was
continued for 26 minutes in order to obtain a maximal skin
vasodilation.
Blood sample collection
About 6ml of venous blood were obtained each time. Fasting blood
glucose, Triglycerides (TG), Total cholesterol (Chol), High-density
lipoprotein (HDL), Low-density lipoprotein (LDL), Blood Urea
Nitrogen (BUN), Creatinine (Cr), hepatic enzymes and Total bilirubin
(Tot. Bil) were determined in all subjects.
Data analysis
All data analyses were performed in a blinded fashion as to the
sequence of the measurement (i.e. Before-fasting, Fasting or Post-
fasting). SkBF was automatically measured (LDI version 5.3D soware,
Moor Instruments Ltd, Axminster, United Kingdom) and was
expressed in Perfusion Unit (P.U.), meaning arbitrary units of blood
ow. e SkBF during BSL scans and hyperemia tests were calculated
and expressed as the percentage of change from the BSL. e Area
under the curve (AUC) was calculated by summing each of the 10
other measures of skin vasodilation in response to Ach and SNP
induced hyperemia. We nally estimated the Ach-AUC to SNP-AUC
ratio (Ach/SNP ratio), in order to determine the relative contributions
of endothelium-dependent over the endothelium-independent
vasodilation. We also analyzed the eect of L-NAME iontophoresis
Before-fasting, during Fasting and Post-fasting periods. e delta
AUC, representing NO-mediated skin thermal hyperemia, was then
calculated as the dierence between the saline and L-NAME AUCs
during the heating-induced hyperemia.
Statistical analysis
All statistical analyses were performed using SPSS soware (PASW
18, Chicago, IL, USA). Data were expressed as mean ± SEM for
quantitative variables and frequencies, and as percentages for
qualitative variables. We used one way ANOVA repeated measure to
determine the dierence in descriptive characteristics and blood
measurement among the 3 periods of test. Categorical variables were
analyzed by Chi-square tests. Student t tests for independent samples
were used to determine dierences in normally distributed data.
Correlation analyses using the Pearson coecient, and a multivariate
correlation were performed to determine the predictability of the
dependent variable from the independent variables by linear
combination. A P value <0.05 was considered statistically signicant.
Results
Subjects’ characteristics (Tables 1 and 2)
e participants were all males and non-smokers with a mean age of
42.4 ± 1.5 years and a BMI of 25.9 ± 1 kg/m² in the fasting group. e
control group was 44.4 ± 0.8 years old and had a BMI of 26.2 ± 0.9
kg/m² (p=NS vs. fasting group). All experiments started at the same
moment of the day (p=NS).
Parameters Before-fasting (n=14) Fasting (n=14) P vs. Before-fasting Post-fasting (n=12) P vs. Before-fasting
Start of experiment (hours) 14:00 ± 0.83 14:32 ± 0.72 NS 14:21 ± 0.90 NS
BMI (Kg/m²) 25.9 ± 1 25.1 ± 1 NS 24.9 ± 0.7 NS
SBp (mmHg) 117 ± 3 104.3 ± 2.8 <0.001 109.2 ± 2.8 NS
DBp (mmHg) 72.5 ± 1.2 67 ± 1.5 <0.01 65.8 ± 2.6 NS
Cholesterol (<190 mg/dL) 180 ± 9.4 189.6 ± 10.4 NS 171.6 ± 9.2 NS
TG (40-150 mg/dL) 106.2 ± 20 119.6 ± 30.4 NS 110 ± 20 NS
HDL-Chol (>40 mg/dL) 51.8 ± 4.2 47.5 ± 3.2 NS 49.7 ± 3.7 NS
LDL-Chol (<115 mg/dL) 107 ± 7.5 118 ± 8 <0.01 99.8 ± 7.3 NS
Fasting Glucose (70-100 mg/dL) 85.6 ± 1.3 93.4 ± 2.5 <0.01 85.7 ± 2 NS
Uric. Acid (2-7.5 mg/dL) 5.7 ± 0.3 5.1 ± 0.3 <0.01 5.2 ± 0.2 <0.05
BUN (15-40 mg/dL) 31.1 ± 1.6 31.3 ± 1.9 NS 27 ± 1.6 <0.05
Cr (0.7-1.2 mg/dL) 0.93 ± 0.04 0.93 ± 0.05 NS 0.94 ± 0.04 NS
Tot. Bil (<1.2 mg/dL) 0.56 ± 0.06 0.46 ± 0.05 NS 0.51 ± 0.07 NS
Phos. Alk (53-128 U/L) 70.4 ± 3 65.7 ± 2.4 <0.05 68 ± 3 NS
ALT (<45 U/L) 26 ± 4.8 25 ± 4.7 NS 26.8 ± 5 NS
AST (<35 U/L) 21.3 ± 1.3 20.3 ± 1.8 NS 22 ± 1.6 NS
Citation: Esmaeilzadeh F, van de Borne P (2016) Does Intermittent Fasting Improve Microvascular Endothelial Function in Healthy Middle-aged
Subjects?. Biol Med (Aligarh) 8: 337. doi:10.4172/0974-8369.1000337
Page 3 of 9
Biol Med (Aligarh), an open access journal
ISSN: 0974-8369
Volume 8 • Issue 6 • 1000337
Days of fasting 0 26 ± 0.5 NA NA NA
Post-fasting days NA 0 NA 34.4 ± 2.5 NA
ALT: Alanine Transaminase; AST: Aspartate Transaminase; BUN: Blood Urea Nitrogen; Chol: Cholesterol; Cr: Creatinine; DBP: Diastolic Blood Pressure; HDL: High
Density Lipoprotein; LDL: Low Density Lipoprotein; NA: Not Applicable; NS: Not Significant; Phos Alk: Phosphatase Alkaline; SBP: Systolic Blood Pressure; TG:
Triglycerides; Tot Bil: Total Bilirubin.
Table 1: Subjects' characteristics in the fasting group.
Changes in biological parameters during fasting (Table 1)
Body mass index did not change during the study (p=NS, Table 1).
Both systolic and diastolic blood pressure (BP) decreased when
compared to the Before-fasting session, albeit signicantly only during
Fasting period (p<0.05). Blood glucose and LDL-cholesterol increased,
while phosphatase alkaline levels decreased, during Fasting (all p<0.05
vs. Before-fasting) but returned to the Before-fasting levels thereaer.
Mean serum TG and BUN did not change during the Fasting period
(both p=NS, vs. Before-fasting). Uric acid was lower during the Fasting
and Post-fasting periods (all p<0.05 vs. Before-fasting). None of these
parameters changed over time in the control group (Table 2).
Parameters 1st test (n=13) 2nd test (n=13) p
Start of experiment (hours) 11:43 ± 0.76 10:51 ± 0.71 NS
BMI (Kg/m²) 26.2 ± 0.9 26.4 ± 0.9 NS
SBp (mmHg) 121.5 ± 5 126.5 ± 5 NS
DBp (mmHg) 80.4 ± 3.7 80.8 ± 3 NS
Cholesterol (<190 mg/dL) 195 ± 7.6 197 ± 6.3 NS
TG (40-150 mg/dL) 90 ± 8.5 92 ± 11.4 NS
HDL-Chol (>40 mg/dL) 57.2 ± 3 56 ± 3 NS
LDL-Chol (<115 mg/dL) 119.6 ± 6.5 122.8 ± 5 NS
Fasting Glucose (70-100 mg/dL) 94.3 ± 4.6 98.2 ± 5.3 NS
Uric. Acid (2-7.5 mg/dL) 5.6 ± 0.2 5.5 ± 0.2 NS
BUN (15-40 mg/dL) 33 ± 1.6 31.8 ± 1.8 NS
Cr (0.7-1.2 mg/dL) 1 ± 0.03 1 ± 0.03 NS
Tot. Bil (<1.2 mg/dL) 0.7 ± 0.08 0.7 ± 0.09 NS
Phos. Alk (53-128 U/L) 57.2 ± 4.4 57.2 ± 4 NS
ALT (<45 U/L) 26 ± 2.8 24.6 ± 2.5 NS
AST (<35 U/L) 22.2 ± 1.7 21 ± 1 NS
Test interval (days) 0 28 ± 1.4 NA
ALT: Alanine Transaminase, AST: Aspartate Transaminase, BUN: Blood Urea Nitrogen, Chol: Cholesterol, Cr: Creatinine, DBP: Diastolic Blood Pressure, HDL: High-
Density Lipoprotein, LDL: Low Density Lipoprotein, NA: Not Applicable, NS: Not Significant, Phos Alk: Phosphatase Alkaline, SBP: Systolic Blood Pressure, TG:
Triglycerides, Tot Bil: Total Bilirubin.
Table 2: Subjects' characteristics in the control group.
Eect of fasting on Ach and SNP skin mediated hyperemia
(endothelial dependent and independent vasodilations)
(Figure 1)
e Ach and SNP baseline skin blood ows did not dier between
the Before-fasting, the Fasting and the Post-fasting measurements (all
p=NS). Intermittent fasting enhanced both Ach and SNP-induced
vasodilatations (both p<0.05). e Ach hyperemic skin reaction was
also enhanced at the Post-fasting session (p<0.05 vs. Before-fasting),
however these changes were not signicant anymore for the SNP
hyperemic skin reaction (Post-fasting vs. Before-fasting, p=NS). As a
result, the Ach/SNP ratio did not change during the time course of the
study (Before fasting: 1.2 ± 0.2 vs. Fasting: 1.1 ± 0.1; and vs. Post-
fasting: 1.4 ± 0.2, all p=NS). All of these parameters did not change
over time in the control group (all p=NS).
Citation: Esmaeilzadeh F, van de Borne P (2016) Does Intermittent Fasting Improve Microvascular Endothelial Function in Healthy Middle-aged
Subjects?. Biol Med (Aligarh) 8: 337. doi:10.4172/0974-8369.1000337
Page 4 of 9
Biol Med (Aligarh), an open access journal
ISSN: 0974-8369
Volume 8 • Issue 6 • 1000337
Figure 1: Ach and SNP induced hyperemia. Eect of intermittent
fasting on endothelial dependent and independent vasodilations,
mediated by Ach iontophoresis (A) and SNP iontophoresis (B) in
the Before-fasting, Fasting, and Post-fasting sessions. BSL indicates
baseline; AUC: Area under the curve; Ach: Acetylcholine; SNP:
Sodium nitroprusside.
Eect of fasting on skin thermal hyperemia (endothelial NO
bioavailability) (Figure 2)
Heating-mediated hyperemia responses in the absence of L-NAME
were not aected in the Fasting and Post-fasting sessions, as compared
to Before-fasting (p=NS). e hyperemic responses aer L-NAME
iontophoresis were reduced during the Fasting and Post-fasting
sessions, when compared to Before-fasting (all p<0.05). As a
consequence, the delta AUC between saline and L-NAME pretreated
skin (Delta Sp-LNAME), which reects the NO-related vasodilation,
increased from 597.4 ± 279% to 1006.3 ± 322.3% (p<0.05, Figure 2B)
during Fasting, and to 821.5 ± 281.3% during Post-fasting (P=NS vs.
Before-fasting, Figure 2C).
Figure 2: Heating induced hyperemia. Eect of intermittent fasting
on heating mediated hyperemia aer skin pretreatment by L-
NAME iontophoresis, represented by Delta AUC SP-LNAME (NO
bioavailability), in the Before-fasting (A), Fasting (B) and Post-
fasting (C) sessions. BSL indicates baseline; AUC: Area under the
curve; Sp-LNAME: Saline-L-N-arginine-methyl-ester; NS: Not
signicant.
Citation: Esmaeilzadeh F, van de Borne P (2016) Does Intermittent Fasting Improve Microvascular Endothelial Function in Healthy Middle-aged
Subjects?. Biol Med (Aligarh) 8: 337. doi:10.4172/0974-8369.1000337
Page 5 of 9
Biol Med (Aligarh), an open access journal
ISSN: 0974-8369
Volume 8 • Issue 6 • 1000337
ese parameters did not change over time in the control group (all
p=NS). Mean serum TG and BUN did not change during the Fasting
period; however individual increases in their levels were adversely
related to NO-related vasodilation enhancement (Figure 3A and B).
Figure 3: Correlation between NO bioavailability and TG&BUN.
Univariate correlations between NO-related vasodilation, serum
triglycerides (A) and blood urea nitrogen (B) at the Fasting period
vs. Before-fasting. AUC indicates Area under the curve; Sp-
LNAME: Saline- L-N-arginine-methyl-ester; NO: Nitric Oxide; TG:
Triglycerides; BUN: Blood Urea Nitrogen.
Discussion
is study tested the hypothesis that a prolonged period of
intermittent fasting improves endothelial function. e main new
ndings of our study are that: 1) Aer almost a month of 19 hours of
daily fasting, both endothelial and non-endothelial microvascular
functions were improved, in spite of momentary rises in blood glucose
and LDL-cholesterol; 2) Increases in serum triglycerides and blood
urea nitrogen hindered these favorable microvascular eects; 3) ese
changes were mostly apparent at the end of the fasting period, with the
exception of the improved hyperemic response to Ach, which persisted
1 month aer fasting cessation, while uric acid and blood urea
nitrogen were lower than Before-fasting; 4) Fasting also induced
temporarily reductions in blood pressure.
is is, to the best of our knowledge, the rst prospective and
controlled study to assess the time dependent eects of intermittent
fasting on microvascular function in humans. e study design of our
study diers markedly from previous numerous uncontrolled trials on
this topic, were some cardiovascular parameters and laboratory
measures were oen recorded before and aer fasting without much
standardization [19,20,34-36]. In our study, measures were carefully
reiterated to determine if the eects of fasting persisted thereaer. A
matched non-fasting group underwent also repeated measurements
over a 1 month period, in order to rule out that non-specic
mechanism, unrelated to fasting, contributed to our ndings. All our
subjects were male, healthy and non-smokers. us, changes in the
menstrual cycle [28-30], as well as in the timing of medication intake
and smoking, cannot explain our observations. We took also great care
to ensure that the time of the day where the experiments were
performed was kept constant throughout the study.
Microvascular function
We performed simultaneously several hyperemic assessments to
improve our understanding of the eects of fasting on microvascular
function. Since these tests elicit vasodilation through dierent
pathways, they provide further insights on the mechanisms involved in
the changes we observed [26]. Aer application of a local anesthetic to
attenuate nonspecic neural mechanisms elicited by the iontophoresis
and heating processes [33], thermal-induced skin vasodilation consists
in a biphasic reaction characterized by an early peak followed by a
plateau [37,38]. is late plateau is chiey mediated by local NO
generation [37,38]. e comparison of this response to the one elicited
by thermal-induced skin vasodilation aer L-NAME iontophoresis,
revealed that fasting enhanced NO-related vasodilation. Because
oxygen-free radicals or, more generally, reactive oxygen species, as well
as reactive nitrogen species, are products of normal cellular
metabolism, fasting-related decreased basal metabolic rate may explain
our results [4,5,21]. e reduced production of superoxide anions
during fasting may prevent rapid NO inactivation and thereby enhance
NO bioavailability [39,40]. No changes occurred when thermal
vasodilatation measures were repeated over the same time in the
matched control group. is is in accordance with a previous study on
the reproducibility and selectivity of thermal-induced skin vasodilation
aer L-NAME iontophoresis [26].
e vascular response to Ach iontophoresis involves endothelium-
derived hyperpolarizing factor (EDHF), NO, and prostaglandins [38].
NO synthase inhibition with L-NAME decreases the cutaneous
heating-induced vasodilation by 20% to 50%, but it reduces the Ach-
induced hyperemia only by 0% to 15% [27]. is may explain the
dierent time courses of changes in the responses to skin vasodilation
in our study, which were signicant for the Fasting and Post-fasting
session for the less NO-dependent Ach response, but achieved
signicance during the Fasting only for the more NO-dependent
thermal response [37,38]. ere are also reasons to believe that
intermittent fasting had a global favorable eect on vascular function
[13,18,23,41], because fasting increased the endothelial-independent
vasodilation in response to SNP. us not only did fasting increase NO
generation, but it also enhanced smooth muscle sensitivity to a NO
donor. Reduction in basal metabolic rate aer fasting could also
account for this latter observation [4,5,21]. Indeed, less superoxide
production may result in fewer reactions with NO to generate
Citation: Esmaeilzadeh F, van de Borne P (2016) Does Intermittent Fasting Improve Microvascular Endothelial Function in Healthy Middle-aged
Subjects?. Biol Med (Aligarh) 8: 337. doi:10.4172/0974-8369.1000337
Page 6 of 9
Biol Med (Aligarh), an open access journal
ISSN: 0974-8369
Volume 8 • Issue 6 • 1000337
cytotoxic peroxynitrite [42]. is may reduce protein nitration, prevent
potassium channel inhibition, lessen vascular cells hyperpolarization,
and thereby improve endothelium-independent relaxation [43,44]. e
fact that the relative contributions of the endothelium-dependent over
the endothelium-independent vasodilation, assessed by the Ach/SNP
ratio, did not change argues also in favor of a global improvement in
endothelium function as a result of intermittent fasting. e Ach
hyperemic skin reaction was enhanced during the Post-fasting session,
when compared to the Before-fasting period. e reason for this
observation is unknown. It could be speculated that the lower uric acid
and BUN during the Post-fasting period indicate that some of the
dietary changes during the fasting period persisted during this latter
period, and played a role in this nding. Changes in uric acid levels
may also have played a role in our ndings throughout the study.
Before fasting uric acid level of 5.7 ± 0.3 mg/dl decreased by
approximately 0.5 mg/dl during the study. Excess uric acid has adverse
endothelial eects, but acts also as a reducing substance [45-49]. In our
study, the acid uric levels remained within the 4.5 mg/dl to 6.2 mg/dl
(or 6.0 mg/dl in the NHANES III cohort [46]) range where the J curve
which relates uric acid concentrations to cardiovascular disease is at
[47,48]. e human vascular smooth and endothelial cells contain
urate transporters (URAT1 [SLC22A12], URATv1/GLUT9 [SLC2A9])
and are sensitive to oxidative stress changes [49]. Depletion of uric acid
due to SLC22A12 (URAT1) loss-of-function mutation alters ow-
mediated dilatation [49]. However, conceivably, less stringent
reductions in uric acid could improve microvascular function.
Metabolic parameters and blood pressure
Mean serum TG and BUN did not change during the Fasting
period; however individual variation in their levels allowed us to show
that their increases were adversely related to an enhanced NO-related
vasodilation. Previous studies have demonstrated that endothelial
function is inversely related to glucose and TG concentrations [50-52].
Although not novel, the relationship between TG level and endothelial
function we observed is worthwhile to mention as it may be regarded
as internal control of scientic data quality. Less known are the
endothelial eects of BUN, also inversely related to endothelial
function improvements in our study. Uremic levels of BUN did not
induce nitric oxide deficiency in rats with normal renal function, but
did so in cultured human and bovine endothelial cells [53]. Elevated
BUN may also reect a negative eect of dehydration on endothelial
function and oxidative stress in our study, since Ramadan fasting
dictates that no liquids are ingested from sunrise to sunset [54,55].
is could participate to the reduction in BP we observed. Moreover,
one month of intermittent fasting decreased markers of sympathetic
activity in animal studies [56]. A similar change in our study would
also translate into a lower BP and an improved endothelial function
[57].
Changes in meal composition occur frequently during Ramadan
fasting [16,58,59]. While one meal is taken aer dawn and the other
before sunset, they contain usually more calories and larger amounts of
sweet and fatty foods. is likely explains the rises in blood glucose
and LDL-cholesterol observed in our study. Albeit these changes,
endothelial function was improved during Fasting. Low carbohydrate
diets result in short-term weight loss and some metabolic benets [60],
but have a deleterious eect on endothelial function [61]. Regarding to
current dietary recommendations, 45-60% of daily energy intake
should be provided from carbohydrates [62]. e carbohydrates
consumed during intermittent fasting, such as dates, honey and home-
made pastry, instead of rened and processed foods and commercial
high glycemic index foods, could play a role in our ndings, since for
vascular protection, carbohydrate quality could be more important
than quantity [63].
Limitations
e sample size of our study is small and our results cannot be
extrapolated to other subjects than the middle-aged non-smoker male
healthy subjects investigated in our study. In mitigation, however, it
should be remembered that the Ramadan fasting started and ended in
all volunteers on the same day. Much larger studies, also using time-
consuming measurements similar to ours, are impractical because all
experimental sessions must occur during a very limited period of time.
Another limitation of our study is that the Ramadan fasting performed
in our study likely diers from the one performed in other regions in
the world, under dierent latitudes, and during dierent seasons.
Shorter or longer intermittent fasting periods may yield dierent
result. Finally, further studies are clearly needed to better understand
the mechanisms involved in the changes in endothelial function we
observed.
Conclusions
Intermittent fasting improved endothelial and non-endothelial
dependent vasodilations and decreased blood pressure. Increased
nitric oxide bioavailability during this period was negatively related to
rises in serum triglycerides and BUN.
Acknowledgments
Sources of Funding: is study was supported by Menarini
(Belgium) (P.v.d.B.), Daichi-Sankyo (Belgium) (P.v.d.B.), and the Astra-
Zeneca (Belgium) and Biotronik (Belgium) chairs for research in
cardiology (P.v.d.B.).
Conict of interest: e authors declare to have no nancial or
personal interests.
Authors’ contributions: EF designed and conducted research; EF
and VBP analyzed data; EF and VBP wrote the paper; EF had primary
responsibility for nal content. All authors read and approved the nal
manuscript.
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