nutrients

Article
Effects of Periodic Fasting on Fatty Liver Index—A
Prospective Observational Study
Stefan Drinda 1,2, *, Franziska Grundler 1,3 , Thomas Neumann 4 , Thomas Lehmann 5 ,
Nico Steckhan 3,6 , Andreas Michalsen 6 and Francoise Wilhelmi de Toledo 1
1 Buchinger Wilhelmi Clinic, 88662 Überlingen, Germany; [email protected] (F.G.);
[email protected] (F.W.d.T.)
2 Clinic St. Katharinental, Department of Rheumatology, 8253 Diessenhofen, Switzerland
3 Charité–Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt–Universität
zu Berlin and Berlin Institute of Health, 10117 Berlin, Germany; [email protected]
4 Division of Rheumatology, Immunology and Rehabilitation, Kantonsspital St. Gallen, 9007 St. Gallen,
Switzerland; [email protected]
5 Institute of Medical Statistics, Computer Sciences and Documentation, Jena University Hospital, 07743 Jena,
Germany; [email protected]
6 Department of Internal and Complementary Medicine, Immanuel Hospital Berlin, 14109 Berlin, Germany;
[email protected]
* Correspondence: [email protected]




Received: 19 September 2019; Accepted: 28 October 2019; Published: 30 October 2019


Abstract: This prospective observational trial investigated effects and safety of periodic fasting in
subjects with and without type 2 diabetes mellitus (T2DM). The primary end point was set as the
change of fatty liver index (FLI) as a surrogate parameter of non-alcoholic fatty liver disease (NAFLD).
Six-hundred and ninety-seven subjects (38 with T2DM) were enrolled. A baseline FLI ≥ 60 (the
threshold for fatty liver) was found in 264 subjects (37.9%). The mean duration of fasting was 8.5 ±
4.0 days (range 6–38). FLI decreased significantly (−14.02 ± 11.67; p < 0.0001), with a larger effect in
individuals with T2DM (−19.15 ± 11.0; p < 0.0001; p = 0.002 compared to non-diabetic subjects). Body
mass index (BMI) decreased by −1.51 ± 0.82 kg/m2 , and 49.9% of the subjects lost ≥5% body weight.
After fasting, nearly half of the 264 subjects with FLI ≥ 60 (highest risk category) shifted to a lower
category. The improvement of FLI correlated with the number of fasting days (r = −0.20, p < 0.0001)
and with the magnitude of BMI reduction (r = 0.14, p = 0.0001). Periodic fasting with concomitant
weight reduction leads to significant rapid improvement of FLI in subjects with and without T2DM.

Keywords: periodic fasting; obesity; overweight; fatty liver index; diabetes

1. Introduction
Non-alcoholic fatty liver disease (NAFLD) is considered one of the most relevant causes of chronic
liver disorders [1], and consists of a disease spectrum including fatty liver, non-alcoholic steatohepatitis,
fibrosis, and liver cirrhosis. The least advanced stage of disease, the non-alcoholic fatty liver (NAFL,
or simple steatosis), is characterized by an excess of fat in the liver and is mostly asymptomatic [2].
The median prevalence of NAFLD is 20%, ranging from 6% to 33% in industrialized countries [3].
The burden of disease is rapidly increasing, mainly due to the rising prevalence of obesity and sedentary
lifestyle, considering that as many as 50% of patients with simple steatosis proceed to develop a
non-alcoholic steatohepatitis (NASH) [4].
NAFLD fuels in a closed loop the epidemics of type 2 diabetes mellitus (T2DM) and metabolic
syndrome [5], almost doubling the risk of developing these disorders [6]. Notably, NAFLD is regarded
as an even stronger predictor for the development of T2DM then waist circumference or obesity [7],

Nutrients 2019, 11, 2601; doi:10.3390/nu11112601 www.mdpi.com/journal/nutrients

Nutrients 2019, 11, 2601 2 of 14

and the degree of histologic liver damage is directly related to the presence of diabetes in morbidly
obese patients [8].
NAFLD is a multi-factorial disease resulting from a complex interaction of environmental and
genetic factors. The most common cause of NAFLD is an altered whole-body energetic homeostasis
resulting from a caloric intake exceeding the caloric expenditure. Energy spill-over is then stored
in the form of non-esterified fatty acids (NEFA) from visceral adipose tissue into ectopic fat depots,
such as the liver, skeletal muscles, and pancreas [9]. NAFLD has been associated with dietary excess of
saturated fatty acids, refined carbohydrates, and fructose [10,11].
The amount of liver fat can be determined by magnetic resonance imaging (MRI) or biopsy, but
it can also be calculated by the fatty liver index (FLI), which has been shown to closely correlate
with the results of the MRI [12]. The FLI is calculated on the basis of body mass index (BMI), waist
circumference, triglycerides (TG), and gamma-glutamyl-transferase (GGT) [13], a calculation that
permits an easy detection of NAFLD and also allows a non-instrumental monitoring of treatment
effects. The FLI score ranges from 0 to 100, with FLI <30 excluding and ≥60 confirming a diagnosis of
fatty liver (including NAFLD).
Several therapeutic strategies can reverse NAFLD [14], but the most important approach is lifestyle
modification with diet and exercise [15,16]. A body weight reduction of 7–10% obtained with energy
restriction and increased physical activity leads to histological improvement of steatosis, inflammation,
and fibrosis [17,18]. Pharmaceutical approaches, in turn, focus directly on hepatic fat accumulation,
anti–inflammatory effects (e.g., oxidative stress alleviation and modulation of tumor necrosis factor),
insulin sensitization, or anti–obesity drugs [14]. Thus far, however, these approaches have shown
rather limited effects on the progression of fatty liver [16,19,20].
While the combination of physical exercise and change of dietary habits is the most effective
intervention for weight loss [21], the translation of long-term lifestyle changes into daily routine
seems to be difficult to achieve [22]. One of the proposed options to ease this difficulty is fasting
therapy, which is the voluntary renouncement of food for a defined period and which results in distinct
metabolic changes, i.e., pronounced lipolysis, decreased insulin secretion, increased insulin sensitivity,
gluconeogenesis, and production of ketone bodies [23]. Periodic fasting, in contrast to intermittent
or Ramadan fasting, is defined as lasting from 2 to 21 days or longer. Periodic fasting has been used
since decades as a treatment option of the metabolic syndrome [23]. Additionally, periodic fasting
does not only induce beneficial effects on lifestyle modification, with a subsequent better adherence to
nutritional recommendations and exercise [24], but can also counteract a prediabetes by restoring beta
cell function and overcoming insulin resistance [25].
To the best of our knowledge, there are no clinical data on the impact of fasting therapy on fatty
liver, although beneficial effects of periodic and intermittent fasting on fatty liver have already been
shown in mice experiments [26]. Currently, it is also unknown if the effects of fasting on fatty liver are
similar in non-diabetic and diabetic individuals. The present study was thus performed to examine the
short-term effects of fasting therapy on FLI in diabetic and non-diabetic subjects, and to investigate the
safety of this approach.

2. Materials and Methods

2.1. Study Design

This study was conducted according to the Declaration of Helsinki and was registered at the
German register of clinical trials (DRKS-ID: 00010111). The study protocol was approved by the
ethics committees of the Charité Medical University of Berlin and the Federal Medical Council of
Baden-Württemberg, Germany.
The subjects agreed to study participation by signing a written informed consent.
The study was designed as a prospective, uncontrolled, observational cohort study. All subjects
were voluntary inpatients at the Buchinger-Wilhelmi Clinic Überlingen (Germany), a medical center
Nutrients 2019, 11, 2601 3 of 14

specialized on periodic fasting. The data presented in this study are part of a larger trial aimed at
investigating safety, health improvement, and well-being in 1422 individuals undergoing periodic
fasting [27].

2.2. Subjects
Study subjects were consecutively recruited between January and October 2016. Inclusion criteria
were age ≥18 years; BMI ≥19 kg/m2 , and a minimum scheduled inpatient stay of 10 days with at least
6 days for periodic fasting. Exclusion criteria were viral or autoimmune liver diseases, cognitive and
psychological disorders, pregnancy or lactation, and inadequate language skills to communicate in
English, French, or German.

2.3. Study Interventions

All participants underwent periodic fasting according to the published guidelines for periodic
fasting therapy [28].
The intervention started with a low-calorie transition day (600 kcal/day mono-diet consisting of
fruits, rice, oat, or vegetables according to patients’ choice). During the fasting period, the subjects
received 250 mL fruit juice or vegetable broth at midday, 250 mL vegetable broth in the evening,
and optional 20 g honey (maximum total energy intake of 250 kcal/day; maximum total carbohydrate
intake 35 g/day). It was recommended to drink at least 2 liters of water/day. The duration of fasting
was adapted to the individual therapeutic goal and tolerance, and was then followed by stepwise
reintroduction of food. The latter consisted of ovo-lacto-vegetarian food increasing from 800 kcal/day
to 1800 kcal/day over at least 3 days. The fasting therapy was accompanied by a physical exercise
program with moderate walking and gymnastics as group activities, paralleled by group sessions in
mindfulness, and relaxation techniques as autogenic training and meditation.

2.4. Outcome Measures

All measurements were done at baseline (day before start of fasting) and at the end of therapy
(day after last fasting day). Anthropometrical measurements (height, weight, and waist circumference)
and blood samples for laboratory assessments were taken between 7.00 and 10.00 a.m. Glucose was
measured using the hexokinase 3 method (Siemens, Erlangen, Germany; coefficients of variation (CV)
11%; normal range 3.9–5.5 mmoL/L); HbA1c by high pressure liquid chromatography (HPLC; Tosoh
Bioscience, Griesheim, Germany, CV 10%, normal <42 mmoL/moL); total cholesterol (TC) by enzymatic
color reaction (Siemens; CV 7%; normal <5.2 mmoL/L); triglycerides (TG) by glycerol phosphate
oxidase (GPO)/Trinder enzymatic color reaction (Siemens; CV 9%; normal <1.7 mmoL/L); high density
lipoprotein (HDL) by a two step catalase–elimination reaction (Siemens; CV 9%; range 1.2–3.1 mmoL/L);
and low density lipoprotein (LDL) by a two–step catalase–elimination reaction (Siemens; CV 6.2%;
normal <4.1 mmoL/L). Glutamate–oxalacetate transferase (GOT), glutamate–pyruvate transferase
(GPT), alkaline phosphatase (AP), and gamma glutamyl transferase (GGT) were measured by
standard methods.
The FLI was calculated as follows: FLI = (e 0.953 × loge (TG) + 0.139 × BMI + 0.718 × loge (GGT)
+ 0.053 × WC − 15.745)/(1 + e 0.953 × loge (TG) + 0.139 × BMI + 0.718 × loge (GGT) + 0.053 × WC −
15.745) × 100 [13].
The study data were analyzed as per protocol analysis.

2.5. Statistical Analysis

The data were presented as means ± standard deviation (SD), if not otherwise indicated.
D’Agostino and Pearson omnibus normality test was used to verify the normal data distribution.
Changes between baseline and end of intervention were compared by the paired test for normally
distributed data and the Wilcoxon matched-pairs signed rank test for non-parametric data. To determine
possible differential effects in diabetic patients, all participants (group all) were clustered in non-diabetic
Nutrients 2019, 11, 2601 4 of 14

subjects (fasting glucose <7 mmoL /L and HbA1c <47 mmoL/moL) and patients with T2DM (fasting
glucose ≥7 and/or HbA1c ≥47 mmoL/moL). Group differences were calculated with the unpaired test
for parametric data or Mann Whitney U test for non-parametric data. Correlations analyses were
performed by the Pearson or Spearman correlation coefficients, depending on the normal distribution
of the data.
A multivariable linear regression model was fitted for FLI after intervention with gender, age,
number of fasting days, GOT, GPT, total cholesterol, diabetes, and baseline FLI as independent variables.
Estimated regression coefficients with 95% confidence intervals (CI) were calculated to evaluate the
influence of the variables.
Additionally, a binary logistic regression model was applied for a positive outcome (defined as
FLI ≤60 after intervention) only for the subjects who had a baseline FLI >60. Independent variables
were gender, age, number of fasting days, GOT, GPT, total cholesterol, diabetes, and baseline FLI.
Receiver-operating curve (ROC) analysis of predicted probabilities was performed, and the area under
curve (AUC) with 95% CI was calculated to measure the accuracy of the model. The discrimination of
this model was assessed by comparing the predicted probabilities with the binary outcomes (FLI ≤ 60)
of each patient in the ROC analysis.
A p value < 0.05 was considered statistically significant. Data analyses were performed by using
the statistical software SPSS version 24 (IBM) and GraphPad Prism 6 (GraphPad Inc.).

3. Results
Of 1500 screened subjects, 741 were included in the study as per inclusion/exclusion criteria.
Of these, 44 dropped out due to: periodic fasting days <6 (n = 27), recently diagnosed liver disease
(n = 12), or non-compliance (n = 5). Thus, 697 subjects completed the study and were analyzed per
protocol. The mean fasting duration was 8.5 ± 4.0 days (range 6–38), with no significant difference (p
= 0.261) between subjects with T2DM (9.3 ± 4.8 days, range 6–31) or without T2DM (8.4 ± 4.0 days,
range 6–38).
Of the 697 study subjects, 38 (5.5%) had T2DM. Of those, 28 were treated with anti–diabetic
medications as follows: metformin: n = 27, dipeptidyl peptidase-4 (DPP4) inhibitors: n = 6, sulfonylurea:
n = 5, sodium-glucose transport 2 (SGLT-2) inhibitors: n = 2, and glitazone: n = 1. Three patients
received a combination of oral anti-diabetics and insulin therapy. One subject received insulin
treatment only.
Treatment for arterial hypertension was recorded in 122 subjects (17.5%), 99/659 in the non-diabetic
subgroup and 23/38 in the T2DM subgroup. Treatment with statins was recorded in 116 subjects
(16.6%), 78/659 in the non-diabetic subgroup and 18/38 in the T2DM subgroup.

3.1. Baseline Characteristics of Study Participants

Baseline characteristics of all study subjects and categorized for the presence or absence of T2DM
are presented in Table 1.
The study population was predominantly female and middle-aged. Patients with T2DM were
older than subjects without diabetes (60.92 ± 9.74 years vs. 54.23 ± 13.46 years, p = 0.002), and their
baseline BMI and waist circumference were significantly higher than in non-diabetic subjects.
The FLI was normal (<30) in 273 subjects (39.2%), intermediate (between ≥30 and <60) in
160 subjects (23.0%), and indicative of fatty liver (≥60) in 264 subjects (37.9%). The subgroup of patients
with T2DM (n = 38) had a significantly higher FLI than subjects without T2DM (78.36 ± 16.97 vs. 44.92
± 31.57, p < 0.001).
Nutrients 2019, 11, 2601 5 of 14

Table 1. Baseline characteristics, comparisons of subjects with and without type 2 diabetes mellitus
(T2DM).

p Value
T2DM No Diabetes All T2DM vs. No
Diabetes
n 38 659 697
Female, n (%) 11 (28.9) 429 (65.1) 440 (63.1)
Age, years 60.92 ± 9.74 54.23 ± 13.46 54.60 ± 13.36 0.0021
BMI, kg/m2 31.79 ± 5.15 27.98 ± 5.25 28.19 ± 5.31 <0.0001
BMI categories
<25 kg/m2 , n (%) 1 (2.6) 206 (79.5) 207 (29.7)
<0.0001
≥25 kg/m2 , n (%) 37 (97.4) 453 (20.5) 490 (60.3)
Height, cm 173.21 ± 0.10 169.23 ± 0.09 169.45 ± 0.09 0.0162
Weight, kg 95.45 ± 17.78 80.46 ± 17.89 81.28 ± 18.19 <0.0001
Waist, cm 107.13 ± 11.32 92.06 ± 15.21 92.88 ± 15.40 <0.0001
Glucose, mmoL/L 8.10 ± 2.40 5.19 ± 0.72 5.35 ± 1.11 <0.0001
HbA1c,
55.2 ± 15.0 35.28 ± 3.94 36.37 ± 6.86 <0.0001
mmoL/moL
GGT, µkat/L 0.75 ± 0.95 0.47 ± 0.53 0.48 ± 0.57 0.0003
GOT, µkat/L 0.46 ± 0.20 0.40 ± 0.21 0.41 ± 0.21 0.0550
GPT, µkat/L 0.63 ± 0.38 0.49 ± 0.35 0.50 ± 0.35 0.0006
AP, µkat/L 1.06 ± 0.31 1.09 ± 0.30 1.09 ± 0.30 0.5164
Cholesterol,
4.83 ± 1.18 5.60 ± 1.18 5.56 ± 1.19 0.0002
mmoL/L
TG, mmoL/L 2.09 ± 0.95 1.52 ± 0.77 1.55 ± 0.80 <0.0001
HDL, mmoL/L 1.17 ± 0.34 1.57 ± 0.47 1.55 ± 0.48 <0.0001
LDL, mmoL/L 3.01 ± 1.02 3.49 ± 1.07 3.46 ± 1.08 0.0083
FLI, points 78.36 ± 16.97 44.92 ± 31.57 46.75 ± 31.86 <0.0001
FLI category
<30 points 1 272 273
≥30–<60 points 4 156 160 <0.0001
≥60 points 33 231 264
BMI: body mass index; Waist: abdominal circumference; GGT: gamma glutamyl transferase; GOT: glutamate
oxalacetate transferase; GPT: glutamate pyruvate transferase; AP: alkaline phosphatase, Cholesterol: total cholesterol;
TG: triglycerides; HDL: high density lipoprotein; LDL: low density lipoprotein. Categorical data were analyzed by
Fischer’s exact test (BMI categories) and chi-square test (fatty liver index (FLI) categories).

3.2. Changes in FLI

Overall, periodic fasting reduced the FLI by a mean of −14.02 ± 11.67 points (p < 0.0001; Figure 1).
As many as 120 of the 264 subjects with a baseline FLI ≥ 60 (high risk category) shifted to a
lower category of FLI risk after therapy. The subgroup of patients with T2DM (n = 38) experienced a
significantly greater FLI reduction (−19.15 ± 11.0 points, p < 0.0001) than non-diabetic subjects (−13.73
± 11.65 points, p < 0.0001; group difference p = 0.002; Figure 2).
 ± 31.57, p < 0.001).

3.2. Changes in FLI

Overall, periodic fasting reduced the FLI by a mean of –14.02 ± 11.67 points (p < 0.0001; Figure
Nutrients 2019, 11, 2601 6 of 14
1).

Figure1.1.Changes
Figure ChangesininFLI
FLIbefore
beforeand
andafter
afterfasting
fastingininpatients
patientswith
withT2DM
T2DM(A),
(A),non-diabetic
non-diabeticsubjects
subjects(B),
(B),
and all subjects (C).
and all subjects (C).
Nutrients 2019, 11, x FOR PEER REVIEW 6 of 14

As many as 120 of the 264 subjects with a baseline FLI  60 (high risk category) shifted to a lower
category of FLI risk after therapy. The subgroup of patients with T2DM (n = 38) experienced a
significantly greater FLI reduction (–19.15 ± 11.0 points, p < 0.0001) than non-diabetic subjects (–13.73
± 11.65 points, p < 0.0001; group difference p = 0.002; Figure 2).

Figure
Figure 2. 2. Frequency
Frequency distribution
distribution ofof
FLIFLI categories
categories before
before and
and after
after therapeutic
therapeutic fasting
fasting inin patients
patients with
with
T2DM (n = 38), in non-diabetic subjects (no T2DM; n = 659), and in all subjects (n
T2DM (n = 38), in non-diabetic subjects (no T2DM; n = 659), and in all subjects (n = 697). = 697).

InInthethenon-diabetic
non-diabetic subgroup,the
subgroup, thenumber
number ofofsubjects
subjects with
with fatty
fatty liver
liver decreased
decreased from
from 231toto
231 124
124
subjects(–46.3%),
subjects (−46.3%), while
while thethe number
number ofof subjects
subjects with
with normalFLI
normal FLI(<30
(<30 points)
points) increased
increased from
from 273273toto
395 subjects (+44.7%). In the T2DM subgroup, the number of patients with fatty liver
395 subjects (+44.7%). In the T2DM subgroup, the number of patients with fatty liver decreased from decreased from
3333toto2121(–36.4%),
(−36.4%), while
while the
the number
number ofof patients
patients with
with normal
normal FLI
FLI increased
increased from1 1toto4.4.An
from Anabsolute
absolute
FLI
FLI reduction
reduction of of >30%
>30% waswas achieved
achieved in 60.9%
in 60.9% of allofsubjects
all subjects whoahad
who had a weight
weight reduction
reduction of 5%of ≥5%
from
from
baseline. baseline.

3.3. Changes in Anthropometric and Metabolic Parameters

3.3. Changes in anthropometric and metabolic parameters
Changes from baseline are shown in Table 2.
Changes from baseline are shown in Table 2.
Periodic fasting induced a significant weight loss in the overall population (−4.37 ± 2.42 kg,
p < 0.001). In the non-diabetic subgroup, the mean changes were −4.31 ± 2.41 kg (p < 0.001), in the
Table 2. Changes from baseline to post fasting, overall and for subjects with and without T2DM.
T2DM subgroup −5.29 ± 2.55 kg (p < 0.001). Half of the subjects (348, 49.9%) lost ≥5% of their body
T2DM (n = 38)
weight. Overall, the BMI decreased by −1.51No diabetes (n 2= 659)
± 0.82 All (n = 697)
kg/m (p < 0.001). In non-diabetic subjects, the BMI
p value
decreased by −1.50Mean ± 0.81
±SD
2
kg/m p(p < 0.001),
value
and in T2DM
Mean ±SD
by −1.75
patients Mean
p value ±SD
± 0.85
p value
< 0.001).
kg/m2 (pT2DM vs.
The waist circumference decreased overall by −5.39 ± 3.28 cm (p < 0.001), in non-diabetic subjects
no diabetes
−5.34kg
byWeight, (p <±0.001),
± 3.27 cm–5.29 2.56 and in T2DM
<0.0001 patients
–4.31 −6.32 ± 3.37
± 2.41 by <0.0001 2.42< 0.001).
cm± (p
–4.37 <0.0001 0.0045
BMI,Fasting
kg/m² plasma –1.75 ± 0.85 <0.0001 –1.50 ± 0.81 <0.0001 –1.51 ± 0.82 <0.0001
glucose and HbA1c levels significantly decreased in all groups. Liver enzymes 0.0213
Waist, cm –6.32 ± 3.37 <0.0001 –5.34 ± 3.27 <0.0001 –5.39 ± 3.28 <0.0001 0.0433
also decreased after the fasting intervention, except for AP in patients with T2DM. The same was found
FLI, points –19.15 ± 11.00 <0.0001 –13.73 ± 11.65 <0.0001 –14.02 ± 11.67 <0.0001 0.0020
for blood
Glucose, lipids. All
mmoL/L blood
–2.69 ± 2.56lipids<0.0001
markedly–0.60
dropped,
± 1.24 except for HDL
<0.0001 cholesterol
–0.72 ± 1.42 and LDL cholesterol
<0.0001 <0.0001
in patients
HbA1c, mmoL/moL with T2DM
–4.43 ±(Table
6.65 2).<0.0001 –1.60 ± 2.91 <0.0001 –1.76 ± 3.28 <0.0001 <0.0001
GGT, µ kat/L –0.18 ± 0.37 <0.0001 –0.12 ± 0.27 <0.0001 –0.12 ± 0.28 <0.0001 0.0694
GOT, µ kat/L 0.29 ± 0.30 <0.0001 0.20 ± 0.28 <0.0001 0.21 ± 0.29 <0.0001 0.0613
GPT, µ kat/L 0.34 ± 0.45 <0.0001 0.18 ± 0.34 <0.0001 0.18 ± 0.35 <0.0001 0.0147
AP, µ kat/L –0.03 ± 0.19 0.3297 –0.04 ± 0.14 <0.0001 –0.04 ± 0.15 <0.0001 0.9886
Cholesterol,
–0.44 ± 1.05 0.0136 –0.66 ± 0.78 <0.0001 –0.64 ± 0.79 <0.0001 0.1858
mmoL/L
Nutrients 2019, 11, 2601 7 of 14

Table 2. Changes from baseline to post fasting, overall and for subjects with and without T2DM.

T2DM (n = 38) No Diabetes (n = 659) All (n = 697)

p Value
Mean ± SD p Value Mean ± SD p Value Mean ± SD p Value T2DM vs. No
Diabetes
Weight, kg −5.29 ± 2.56 <0.0001 −4.31 ± 2.41 <0.0001 −4.37 ± 2.42 <0.0001 0.0045
BMI, kg/m2 −1.75 ± 0.85 <0.0001 −1.50 ± 0.81 <0.0001 −1.51 ± 0.82 <0.0001 0.0213
Waist, cm −6.32 ± 3.37 <0.0001 −5.34 ± 3.27 <0.0001 −5.39 ± 3.28 <0.0001 0.0433
FLI, points −19.15 ± 11.00 <0.0001 −13.73 ± 11.65 <0.0001 −14.02 ± 11.67 <0.0001 0.0020
Glucose,
−2.69 ± 2.56 <0.0001 −0.60 ± 1.24 <0.0001 −0.72 ± 1.42 <0.0001 <0.0001
mmoL/L
HbA1c,
−4.43 ± 6.65 <0.0001 −1.60 ± 2.91 <0.0001 −1.76 ± 3.28 <0.0001 <0.0001
mmoL/moL
GGT, µkat/L −0.18 ± 0.37 <0.0001 −0.12 ± 0.27 <0.0001 −0.12 ± 0.28 <0.0001 0.0694
GOT, µkat/L 0.29 ± 0.30 <0.0001 0.20 ± 0.28 <0.0001 0.21 ± 0.29 <0.0001 0.0613
GPT, µkat/L 0.34 ± 0.45 <0.0001 0.18 ± 0.34 <0.0001 0.18 ± 0.35 <0.0001 0.0147
AP, µkat/L −0.03 ± 0.19 0.3297 −0.04 ± 0.14 <0.0001 −0.04 ± 0.15 <0.0001 0.9886
Cholesterol,
−0.44 ± 1.05 0.0136 −0.66 ± 0.78 <0.0001 −0.64 ± 0.79 <0.0001 0.1858
mmoL/L
TG, mmoL/L −0.78 ± 0.96 <0.0001 −0.43 ± 0.69 <0.0001 −0.44 ± 0.71 <0.0001 0.0057
HDL, mmoL/L −0.07 ± 0.27 0.0905 −0.24 ± 0.26 <0.0001 −0.23 ± 0.27 <0.0001 <0.0001
LDL, mmoL/L −0.20 ± 1.03 0.2327 −0.31 ± 0.79 <0.0001 −0.30 ± 0.81 <0.0001 0.6141
BMI = body mass index; Waist = abdominal circumference; GGT = gamma glutamyl transferase; GOT = glutamate
oxalacetate transferase; GPT = glutamate pyruvate transferase; AP = alkaline phosphatase, Cholesterol = total
cholesterol; TG = triglycerides; HDL = high density lipoprotein; LDL = low density lipoprotein. Data were analyzed
by a paired t–test.

3.4. Correlations Analyses

The results of the correlation analyses are shown in Table 3.

Table 3. Correlation analyses, overall and for subjects with and without T2DM.

T2DM (n = 38) No Diabetes (n = 659) All (n = 697)

r p Value r p Value r p Value
FLI vs.
−0.42 0.0091 −0.18 <0.0001 −0.20 <0.0001
Fasting days
FLI vs. BMI 0.32 0.0474 0.27 <0.0001 −0.14 0.0001
FLI vs. WC 0.39 0.0165 0.28 <0.0001 0.29 <0.0001
FLI vs. GGT 0.22 0.1907 0.48 <0.0001 0.47 <0.0001
FLI vs. GOT 0.18 0.2716 −0.10 0.0102 −0.10 0.0120
FLI vs. GPT 0.18 0.2802 −0.02 0.6154 −0.02 0.5673
FLI vs. AP 0.35 0.0322 0.17 <0.0001 0.18 <0.0001
FLI vs.
0.33 0.0451 0.30 <0.0001 0.29 <0.0001
Cholesterol
FLI vs. TG 0.23 0.1576 0.63 <0.0001 0.62 <0.0001
FLI vs. fG 0.03 0.8563 0.07 0.0942 −0.02 0.6890
FLI vs.
0.14 0.4096 0.04 0.3186 0.06 0.0891
HbA1C
BMI = body mass index; WC = waist circumference; GGT = gamma glutamyl transferase; GOT = glutamate
oxalacetate transferase; GPT = glutamate pyruvate transferase; AP = alkaline phosphatase, Cholesterol = total
cholesterol; TG = triglycerides; fG = fasting glucose. r = Pearson’s correlation coefficient for parametric data or
Spearman’s correlation coefficient for non-parametric data.
Nutrients 2019, 11, 2601 8 of 14

The decreases in FLI induced by fasting correlated with the length of the fasting, in the overall
group (r = −0.20; p < 0.0001) as well as in the subgroups of non-diabetic subjects (r = −0.18; p < 0.0001)
and T2DM patients (r = −0.36; p = 0.0262; Figure 3).
Likewise, changes in FLI significantly correlated with the decrease of body weight and waist
circumference (Table 3, Figure 4).
There were no significant correlations between changes in FLI and fasting plasma glucose or
Nutrients 2019,
11,11, x FOR PEER REVIEW 8 of
1414
HbA1c levels
Nutrients (Table
2019, 3). PEER
x FOR REVIEW 8 of

Figure 3. Correlation of FLI changes with fasting duration in patients with T2DM (A), non-diabetic
Figure
Figure 3. 3. Correlation
Correlation of of FLI
FLI changes
changes with
with fasting
fasting duration
duration inin patients
patients with
with T2DM
T2DM (A),
(A), non-diabetic
non-diabetic
subjectssubjects
(B), and
(B),all subjects
and (C); (C);
andand
with changes ofofBMI in patients with T2DM(D),
(D),non-diabetic
non-diabetic
subjects (B), and allall subjects
subjects (C); and with
with changes
changes BMI
of BMI inin patients
patients with
with T2DM
T2DM (D), non-diabetic
subjectssubjects
(E), and(E),alland
subjects (F). (F).
all subjects
subjects (E), and all subjects (F).

Figure 4. Reduction
Figure
Figure 4. 4.
of of
Reduction
FLI
Reduction of
FLI
ininrelation
FLI in to
relation
relation
percent
to to percent
percent
changes
changes
changes
inbody
inin
body
body mass
mass
mass
index
index
index
(BMI).
(BMI).
(BMI).

Predictorsofofchanges
Predictors changesininFLI
FLIwere
wereanalyzed
analyzedinina amultivariate
multivariatelinear
linearregression
regressionmodel
modelwithwith
gender, age, number of fasting days, GOT, GPT, total cholesterol, diabetes, and baseline FLI asas
gender, age, number of fasting days, GOT, GPT, total cholesterol, diabetes, and baseline FLI
independentvariables.
independent variables.FLI
FLIdecreased
decreasedononaverage
averagebyby0.48
0.48points
pointswith
witheach
eachadditional
additionalfasting
fastingday
day
(regression coefficient beta: –0.48, 95% CI –0.665 to –0.295, p < 0.001). FLI reduction was also nearly 4 4
(regression coefficient beta: –0.48, 95% CI –0.665 to –0.295, p < 0.001). FLI reduction was also nearly
Nutrients 2019, 11, 2601 9 of 14

Predictors of changes in FLI were analyzed in a multivariate linear regression model with gender,
age, number of fasting days, GOT, GPT, total cholesterol, diabetes, and baseline FLI as independent
variables. FLI
Nutrients 2019, 11,decreased on average by 0.48 points with each additional fasting day (regression
x FOR PEER REVIEW 9 of 14
coefficient beta: −0.48, 95% CI −0.665 to −0.295, p < 0.001). FLI reduction was also nearly 4 points
pointsfor
higher higher
malesfor males95%
(−3.94, (–3.94, 95% CIto–5.780
CI −5.780 −2.10,top –2.10, p <than
< 0.001) 0.001)
forthan for females.
females. GOT95%
GOT (10.61, (10.61, 95%to
CI 4.77 CI
4.77 to 16.46, p < 0.001), baseline FLI (–0.139, 95% CI –0.169 to –0.110, p < 0.001), and total
16.46, p < 0.001), baseline FLI (−0.139, 95% CI −0.169 to −0.110, p < 0.001), and total cholesterol (0.027, cholesterol
(0.027,
95% 95% CIto
CI −0.044 –0.044
−0.010,to –0.010, p = were
p = 0.002) 0.002)also
were also significant
significant predictors
predictors in thisin this model.
model. A ROC A curve
ROC curve
was
was calculated to test the performance of this model (input variables: gender, age,
calculated to test the performance of this model (input variables: gender, age, number of fasting days, number of fasting
days,GPT,
GOT, GOT, GPT,
total total cholesterol,
cholesterol, diabetes,diabetes, and baseline
and baseline FLI) in discriminating
FLI) in discriminating the capability
the capability to shift
to shift from a
from
FLI > 60a FLI > 60≤to
to a FLI 60adueFLIto≤ periodic
60 due to periodic
fasting fasting
(Figure (Figure
5). With this5). Withthe
model, thisability
model, the ability to
to discriminate
discriminate
subjects proved subjects
relativelyproved
highrelatively high (AUC
(AUC = 0.947, 95% CI= 0.922–0.971,
0.947, 95% CIp 0.922–0.971,
< 0.001). p < 0.001).

Figure 5. Receiving operator characteristic (ROC) curves of gender, age, number of fasting days, and
Figure 5. Receiving operator characteristic (ROC) curves of gender, age, number of fasting days, and
baseline parameters (GOT, GPT, total cholesterol, diabetes status, and FLI) for the prediction of FLI ≤60
baseline parameters (GOT, GPT, total cholesterol, diabetes status, and FLI) for the prediction of FLI
after fasting intervention.
≤60 after fasting intervention.
3.5. Safety
3.5. Safety
Adverse events were reported in 10 study subjects (1.4%). None of the events met the criteria
Adverse events
of seriousness. were reported
The following eventsinoccurred
10 study insubjects (1.4%).
more than oneNone of theeczema
subject: events (nmet= the criteria
3) and mildof
seriousness. The
hyponatremia following
(n = 2). eventsevents
The following occurred in more
occurred in onethan one each:
subject subject: eczema (n
self-limiting = 3) and mild
supraventricular
hyponatremia (n = 2). The following events occurred in one
tachycardia, intermittent self-limiting paroxysmal atrial fibrillation, mild hypokalemia, bleeding subject each: self-limiting
gums,
common cold, dizziness, mild hypoglycemia, intermittent visual disorders, and headache. None ofmild
supraventricular tachycardia, intermittent self-limiting paroxysmal atrial fibrillation, the
hypokalemia,
subjects bleeding
discontinued the gums,
fasting common
because ofcold, dizziness, mild hypoglycemia, intermittent visual
the events.
disorders,
A dailyand headache. None
self-reporting form for of the subjects
feelings discontinued
of hunger showedthe thatfasting
363 ofbecause of the events.(62.7%;
the 579 respondents
A dailywith
29 of whom self-reporting
T2DM) did formnotfor feelings ofrelevant
experience hunger showed
hunger that 363 the
during of the 579 respondents
fasting (62.7%;
therapy, whereas
29 of whom with T2DM) did not experience relevant hunger during the
217 subjects (37.3%) had at least one episode of hunger. No one indicated to be hungry every day fasting therapy, whereas 217
subjects (37.3%)
during fasting. had at least one episode of hunger. No one indicated to be hungry every day during
fasting.
4. Discussion
4. Discussion
Diet interventions are well-established strategies to reduce body weight and improve glucose
Diet interventions
metabolism, however noare well-established
evidence exists about strategies
the effectsto ofreduce
periodicbody weight
fasting onand improve
NAFLD. Thisglucose
study
metabolism, however no evidence exists about the effects of periodic fasting
is the first to show the beneficial effects of periodic fasting on fatty liver. The results of our on NAFLD. Thisstudy
study
is the firstthe
supported to show the beneficial
hypothesis that FLI,effects of periodic
a surrogate fastingofon
parameter fatty
fatty liver.
liver, The results decreases
significantly of our studyin
individuals with and without T2DM after a fasting intervention of at least 6 days. The prospectivein
supported the hypothesis that FLI, a surrogate parameter of fatty liver, significantly decreases
individuals
study designwith and without
according T2DM afterprotocol
to a standardized a fastingwith
intervention of at leastfasting
a well-established 6 days.technique
The prospective
and a
study
close designmonitoring
clinical according wasto a astandardized
strength of this protocol
study. with a well-established fasting technique and a
close clinical
Weight monitoring
reduction andwas a strength of
improvement of fatty
this study.
liver indicators are known to be interrelated [17,18].
Weight
Our results reduction
indicate thatandeven improvement of fatty liver
a modest reduction of BMIindicators
improved are surrogate
known to markers
be interrelated
of fatty[17,18].
liver.
Our results indicate that even a modest reduction of BMI improved surrogate markers of fatty liver.
Indeed, in nearly half of the subjects in the highest risk category (FLI > 60), a BMI reduction of less
than 5% was sufficient to induce a shift to a lower risk category.
In most patients, NAFLD is associated with features of metabolic syndrome, central obesity,
elevated blood pressure, dyslipidemia, hyperglycemia, and insulin resistance [29]. Although these
pathologies can be addressed by lifestyle interventions, in daily life the adherence to the necessary
Nutrients 2019, 11, 2601 10 of 14

Indeed, in nearly half of the subjects in the highest risk category (FLI > 60), a BMI reduction of less
than 5% was sufficient to induce a shift to a lower risk category.
In most patients, NAFLD is associated with features of metabolic syndrome, central obesity,
elevated blood pressure, dyslipidemia, hyperglycemia, and insulin resistance [29]. Although these
pathologies can be addressed by lifestyle interventions, in daily life the adherence to the necessary
lifestyle changes is poor, resulting in suboptimal outcomes. In contrast, in pragmatic programs there
is a greater benefit in a more substantial weight loss, particularly at early stages of the intervention
period [30]. Hence, periodic fasting can significantly reduce weight, and this effect can be maintained
over time [31].
There are several concerns about the adverse effects of fasting. Several non-fatal (e.g., headache) and
rarely fatal (e.g., ventricular arrhythmia) events have been reported [32]. In contrast, no severe adverse
events were found in a cohort of 1422 subjects treated with a periodic fasting lasting 4–21 days, [27].
Michalsen et al. evaluated the acceptance, safety, effects on health-related outcomes, and lifestyle
adherence of fasting therapy in different chronic internal diseases [24]. They found no serious adverse
events throughout their study. Our study supports the hypothesis that fasting therapy provided in a
controlled clinical setting is a safe intervention.
There is a large body of evidence on the beneficial effects of carbohydrate restriction and
hypocaloric diets on NAFLD [33]. It has also been shown that-at equal levels of weight reduction-a
carbohydrate-restricted diet leads to a significantly greater intrahepatic triglyceride reduction than
low-calorie diet alone [34]. The metabolic advantage of carbohydrate restriction appears to be related
to a more pronounced lipid oxidation and enhanced ketogenesis. In recent years, intermittent and
periodic fasting has gained popularity as an alternative to continuous caloric restriction. In addition to
the weight loss effects, periodic fasting is associated with several metabolic benefits, including the
improvement of lipid profiles [27]. This has been also shown for intermittent fasting, e.g., Ramadan
fasting [35]. The key mechanism responsible for many of these beneficial effects is the metabolic switch
from the utilization of glycogenolysis-derived glucose to fatty acids and fatty acid-derived ketones, i.e.,
a fundamental switch from lipid synthesis and fat storage to mobilization of fat in the form of free
fatty acids and fatty acid-derived ketones. This occurs between 12 and 36 hours after cessation of food
consumption [36]. Hyperinsulinemia suppresses ketogenesis and therefore prolongs the time to switch
in cases of obesity, insulin resistance, and T2DM [37]. Although there is some evidence of impaired
ketogenesis during the progression of liver disease to steatohepatitis, therapies that increase hepatic
ketogenesis are expected nonetheless to ameliorate NAFLD [38,39].
Our data provided first evidence that periodic fasting leads to a clearance of liver fat: fasting
significantly reduced FLI and increased the proportion of patients without NAFLD (FLI < 30 units;
Figure 2). The effects of fasting therapy were stronger in males and in individuals with higher baseline
FLI, higher GOT, and higher cholesterol levels. Each additional fasting day further decreased the FLI.
The binary logistic regression showed that every day of fasting increase by 40% the chance to improve
a manifest fatty liver (FLI > 60) and switch to a lower category of risk. This implies that the duration of
fasting must be sufficient to influence fatty liver positively. This should be taken into account when
periodic fasting is considered as treatment for NAFLD.
As already mentioned, insulin resistance, T2DM, and the development of NAFLD are closely
associated conditions. Taylor et al. have shown that remission of T2DM requires a decrease of liver
fat [40]. Patients with T2DM tend to have higher FLI scores, but in this study, we could demonstrate
that the fasting intervention was equally effective in T2DM patients and in non-diabetic subjects in
terms of FLI, although the improvement of other parameters (e.g., HDL, LDL, and AP) was not as
complete. These results are in line with a previous study on periodic fasting in T2DM [31].
Liver enzymes, insulin resistance, and cholesterol levels are related in NAFLD [41,42]. Our results
supported a correlation between changes in FLI and changes in liver enzymes (GGT, and GOT) and
lipid parameters (TG) after fasting intervention, although this was limited to subjects without T2DM.
Nutrients 2019, 11, 2601 11 of 14

The intervention was well tolerated and adverse events were rare. Interestingly, the majority
of participants did not feel hungry during fasting, as reported also in other studies [32]. Periodic
fasting leads to significant weight loss, reduction of BMI and waist circumference. These observations
are interpreted as positive effects, considering that at baseline the participants were pre-obese
(BMI > 25 kg/m2 ; overall and in the non-diabetic subgroup) or obese (BMI > 30 kg/m2 ; in the T2DM
subgroup). Both pre-obesity and obesity are regarded as general health risk factors [43].
Our observational study has some limitations. The analyses were carried out as pre- to post
intervention changes, without a control group, and were focused on FLI as surrogate parameter for
NAFLD, which have been shown to correlate with MRI assessments of fatty liver [12]. MRI assessments
are regarded as the gold standard for the diagnostics of NAFLD, but this type of external control was
not feasible in this large observational study. There was also no significant difference in fasting duration
between diabetic and non-diabetic patients, but we did not match the groups before intervention
because of the very different numbers of individuals in each group. Finally, we could not collect data
on long-term effects after the fasting intervention, therefore a prediction of sustainability of the fasting
effects on hepatic changes is not possible. Lean et al. reported that at 12 months almost half of T2DM
patients achieved remission to a no-diabetic state and off antidiabetic drugs after intervention with
3–5 months of formula diet (825–853 kcal/day). It is reasonable to expect that periodic fasting gains
similar effects in shorter time with good tolerance [44].
In conclusion, periodic fasting can be regarded as an easily realizable, well-tolerated,
non-pharmaceutical intervention, which effectively reduces the FLI. This effect was seen in individuals
with and without T2DM. Further studies with a control group and long-term follow-up are needed to
better characterize the positive effects of periodic fasting on fatty liver and the adaptations in glucose
and lipid metabolism.

Author Contributions: S.D., F.G., A.M., and F.W.d.T. designed the study; F.G. coordinated the study and collected
the data, S.D., T.N., T.L. and N.S. interpreted data; S.D. and T.N. and F.G. drafted the manuscript; A.M. and F.W.d.T.
reviewed and edited the manuscript; all authors approved the final version of the manuscript.
Funding: The study was funded by Amplius GmbH, Überlingen, Germany. This company develops a research
department for the Buchinger Wilhelmi Clinics Überlingen and Marbella. The funders had no role in study design,
data collection and analysis, decision to publish, or preparation of the manuscript.
Acknowledgments: The authors thank Ernesta Palombo–Kinne (Jena) for critical revision of the manuscript.
Conflicts of Interest: F.W.d.T. is member of the Directory Board of the Buchinger Wilhelmi Clinic where the study
was performed. Amplius GmbH is a company that conceives, coordinates and develops fasting research. A.M. is
a consultant at Buchinger Wilhelmi Clinic and receives financial compensation for this role from Amplius GmbH.
This does not alter the authors’ adherence to Nutrients policies on sharing data and materials. S.D., T.N., T.L., F.G.
and N.S. have no conflicts to declare.

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