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RESEARCH ARTICLESafety, health improvement and well-beingduring a 4 to 21-day fasting period in anobservational study including 1422 subjectsFrançoise Wilhelmi de Toledo ID1 *, Franziska Grundler1,2 , Audrey Bergouignan3,4,5,6,Stefan Drinda1, Andreas 111111a11111111111 Buchinger Wilhelmi Clinic, Überlingen, Germany, 2 Charité–Universitätsmedizin Berlin, corporate memberof Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany,3 Division of Endocrinology, Metabolism, and Diabetes and Anschutz Health and Wellness Center, Universityof Colorado, School of Medicine, Aurora, Colorado, United States of America, 4 Division of GeriatricMedicine, University of Colorado, School of Medicine, Aurora, Colorado, United States of America, 5 InstitutPluridisciplinaire Hubert Curien, Université de Strasbourg, CNRS, Strasbourg, France, 6 UMR 7178 CentreNational de la Recherche Scientifique (CNRS), Strasbourg, France, 7 Institute of Social Medicine,Epidemiology and Health Economics, Charité–Universitätsmedizin Berlin, corporate member of FreieUniversität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany,8 Department of Internal and Integrative Medicine, Immanuel Krankenhaus Berlin, Berlin, Germany These authors contributed equally to this work.* [email protected] ACCESSCitation: Wilhelmi de Toledo F, Grundler F,Bergouignan A, Drinda S, Michalsen A (2019)Safety, health improvement and well-being duringa 4 to 21-day fasting period in an observationalstudy including 1422 subjects. PLoS ONE 14(1):e0209353. : Massimiliano Ruscica, Università degliStudi di Milano, ITALYReceived: June 18, 2018Accepted: December 3, 2018Published: January 2, 2019Copyright: 2019 Wilhelmi de Toledo et al. This isan open access article distributed under the termsof the Creative Commons Attribution License,which permits unrestricted use, distribution, andreproduction in any medium, provided the originalauthor and source are credited.Data Availability Statement: All relevant data arewithin the paper and its Supporting Informationfiles.Funding: The study was financed by AmpliusGmbH, Überlingen, Germany. This company hasthe task to develop a research department for theBuchinger Wilhelmi Clinics Überlingen andMarbella who are the funders. The funders had norole in study design, data collection and analysis,decision to publish, or preparation of theAbstractOnly few studies document longer periods of fasting in large cohorts including non-obeseparticipants. The aim of this study was to document prospectively the safety and anychanges in basic health and well-being indicators during Buchinger periodic fasting within aspecialised clinic. In a one-year observational study 1422 subjects participated in a fastingprogram consisting of fasting periods of between 4 and 21 days. Subjects were grouped infasting period lengths of 5, 10, 15 and 20 2 days. The participants fasted according to theBuchinger guidelines with a daily caloric intake of 200–250 kcal accompanied by a moderate-intensity lifestyle program. Clinical parameters as well as adverse effects and well-beingwere documented daily. Blood examinations before and at the end of the fasting period complemented the pre-post analysis using mixed-effects linear models. Significant reductions inweight, abdominal circumference and blood pressure were observed in the whole group(each p 0.001). A beneficial modulating effect of fasting on blood lipids, glucoregulation andfurther general health-related blood parameters was shown. In all groups, fasting led to adecrease in blood glucose levels to low norm range and to an increase in ketone bodies levels (each p 0.001), documenting the metabolic switch. An increase in physical and emotional well-being (each p 0.001) and an absence of hunger feeling in 93.2% of the subjectssupported the feasibility of prolonged fasting. Among the 404 subjects with pre-existinghealth-complaints, 341 (84.4%) reported an improvement. Adverse effects were reported inless than 1% of the participants. The results from 1422 subjects showed for the first timethat Buchinger periodic fasting lasting from 4 to 21 days is safe and well tolerated. It led toenhancement of emotional and physical well-being and improvements in relevant cardiovascular and general risk factors, as well as subjective health complaints.PLOS ONE https://doi.org/10.1371/journal.pone.0209353 January 2, 20191 / 23

Safety, health and well-being during periodic fastingmanuscript. No additional external funding receivedfor this study.Competing interests: FWT is member of theDirectory Board of the Buchinger Wilhelmi Clinic(BWC), where the study was performed. Asmanaging director of Amplius GmbH, FWTexecutes the scientific leadership at BWC. AmpliusGmbH is a company that conceives, coordinatesand develops fasting research on behalf of BWC.FG is currently employed, and SD was formerlyemployed, at BWC. AM is a consultant at BWC andreceives financial compensation for this role fromAmplius GmbH. This does not alter the authors’adherence to PLOS ONE policies on sharing dataand materials.IntroductionThere are a growing number of recent publications on intermittent fasting (IF), generally lasting 16 to 48 hours, and calorie restriction (CR). Periodic fasting (PF), recently defined as lasting from 2 to as many as 21 or more days, is less studied in humans, especially for periodslonger than 4 days [1, 2]. Results show that lifespan and healthspan are prolonged in severalanimal models by fasting and CR [2–7] and that parameters of age-related diseases areimproved in humans [5, 8, 9]. Fasting leads to pronounced metabolic changes: The shift fromcarbohydrates and glucose to fatty acids and ketones as the major cellular fuel source for bodyand brain seems to play a key role. It has recently been referred to as intermittent metabolicswitching (IMS) and glucose-to-ketone (G-to-K) switch. The reverse step–ketone-to-glucose(K-to-G) switch–happens upon refeeding [2]. The G-to-K switch includes reduction in bloodglucose, insulin and IGF-1 levels, depletion or reduction of glycogen stores, and an increase inlipolysis and ketogenesis [2, 5, 10]. Fasting has been shown to induce differential cellular stressresistance [11] and autophagy [12, 13], as well as triggering the synthesis of detoxificationenzymes [9, 14]. Fasting seems to modify the intestinal microbiome [9, 15]. It also leads tochanges in the intestinal mucosal walls in rats [16] and to pronounced neuroendocrine adaptation processes [2, 17]. Finally, in the K-to-G switch, fasting has been found to activate stemcells and multiple system regeneration in the refeeding period [4, 18, 19] and to increase themitochondrial biogenesis in neurons and other body cells [2, 9].Long periods of fasting, lasting several days to several weeks, are physiologic, e.g. duringseasons of low sun exposure, and are still part of the life of most animals [20, 21] as well as ofhumans living without food conservation technologies [22].Fasting periods with various patterns are found in most religions [23]. For instance, Ramadanintermittent fasting was linked with improvements in cardiometabolic risk factors [24]. Furthermore, morbid obesity and associated diseases were treated in the 1960s with long periods of fasting that were termed the “zero calorie diet” [25, 26]. In exceptional circumstances these periodscould last up to 249 days or more [27, 28]. A medical program of periodic fasting developed bythe German physician Otto Buchinger to treat obesity and metabolic and inflammatory pathologies is well-known in central Europe [29–31]. The therapeutic effects of Buchinger periodic fastingare documented in small studies on overweight [32], blood pressure [33], metabolic syndrome[34], fibromyalgia [35], chronic pain syndromes [36] and the enhancement of quality of life [37].The effects of repeated cycles of Buchinger fasting have also been reported [38, 39].We are not aware of large studies on PF including normal weight or moderately obese subjects and focused on safety and tolerability. In the present observational study, we documentedprospectively the safety, general health-related outcomes and well-being of 1422 subjects. Theyfasted for periods between 4 to 21 days under medical supervision according to the Buchingerfasting program, as described in peer-reviewed guidelines [31]. The fasting took place in a facility specialized in therapeutic fasting, the Buchinger Wilhelmi clinic (BWC) in Germany. Theprotocol involved daily clinical monitoring, intake of 2–3 L of water per day and 250 kcal offood, as well as a multi-disciplinary program including health education and physical activity.The aim of this study was to assess for the first time prospectively the safety, therapeutic efficiency and effects on well-being of Buchinger periodic fasting in a large cohort.Materials and methodsEthicsThis observational and prospective study was approved by the medical council of Baden-Württemberg and the Ethics Committee of the Charité-University Medical Center, BerlinPLOS ONE https://doi.org/10.1371/journal.pone.0209353 January 2, 20192 / 23

Safety, health and well-being during periodic fasting(application number: EA4/054/15) on 5 May 2015. The study protocol was registered in theGerman Clinical Trials Register (DRKS-ID: DRKS00010111). The authors confirm that allongoing and related trials for this intervention are registered. At the time of obtaining the ethical approval by the German authorities it was not mandatory to register an observationalstudy. Only randomized clinical trials were clearly recommended to register. Nevertheless, wedecided to register this observational study (on 3 June 2016).Participants were enrolled after giving their written informed consent between 1 Januaryand 31 December 2016. The study was conducted in the BWC in Überlingen (Germany) inaccordance with the principles of the Declaration of Helsinki. The follow-up was completedbetween 26 January 2016 and 18 December 2017.ParticipantsOur study on Buchinger fasting during periods of 4 to 21 days included 1422 subjects. Theywere selected out of a total of 3929 subjects who were admitted to the BWC and fulfilled thefollowing criteria: they had a clinic stay of at least 10 nights and signed the informed writtenconsent at the beginning of the inpatient stay after confirming that they would not participatein another study. Subjects were aged between 18–99 years and had no predefined contraindication to Buchinger fasting (e.g. cachexia, anorexia nervosa, advanced kidney, liver or cerebrovascular insufficiency, dementia or other severely debilitating cognitive disease and no existingpregnancy or lactation period) [31]. Furthermore, blood must have been collected on the precise days defined in the protocol (S2 and S3 Text). We excluded subjects who were prescribedother diets than fasting according to predefined criteria as well as those who could not followthe study procedures due to an inability to speak German, English or French. A flow chartreflecting the selection procedure is given in Fig 1.The subjects came voluntarily to the BWC for preventive or therapeutic reasons. Theyselected established programs of 5, 10, 15 and 20 fasting days, which are reflected in the 4groups (F5d, F10d, F15d, F20d). The main personal intentions for the fasting interventionwere reduction of cardiovascular risk factors, weight loss in case of obesity and relief of generalhealth problems such as inflammatory diseases, distress and exhaustion. In case of prescribeddrug intake, the dosage was adapted during the stay by the 8 physicians of the clinic, whoexamined all participants 2 to 3 times per week. The subjects had a wide diversity of nationaland cultural backgrounds. The majority of them came from upper social classes and had higheducation levels.Fasting programAll subjects fasted according to the guidelines of the Buchinger fasting therapy [31] underdaily supervision of nurses and specialized physicians. On the day before the beginning of thefast, the participants were given a 600 kcal vegetarian diet divided into 3 meals of either riceand vegetables or fruits, according to individual preference. To initiate the fasting period, theintestinal tract was emptied through the intake of a laxative (20–40 g NaSO4 in 500 ml water).During fasting all subjects were asked to drink 3 L of water or non-caloric herbal teas dailywith an optional portion of 20 g honey. Additionally, an organic freshly squeezed fruit or vegetable juice (250 ml) was served at noon and a vegetable soup (250 ml) in the evening, leadingto an average total calorie intake of 200–250 kcal and 25–35 g of carbohydrates per day. At thebeginning of the fasting period the subjects entered a program of light physical exercise alternating with rest and individual mild non-physical treatments like hydrotherapy or physiotherapy. The exercise program consisted of light to moderate intensity outdoor walks and groupgymnastics. The whole program was led by certified trainers. During the fasting period anPLOS ONE https://doi.org/10.1371/journal.pone.0209353 January 2, 20193 / 23

Safety, health and well-being during periodic fastingFig 1. Flow chart of the selection procedure of study 0209353.g001enema or, if preferred by the patient, a mild laxative was applied every second day in order toremove intestinal remnants and desquamated mucosal cells. On the last day of fasting, foodwas stepwise reintroduced during an average of 4 days, with an ovo-lacto-vegetarian organicdiet progressively increasing from 800 to 1600 kcal/day.MeasurementsTo document safety as well as health benefits and well-being during a prolonged periodic fasting program, we performed the predefined following measurements at baseline (pre-) and atthe completion of fasting (post-). Before starting the fast all subjects went through a thoroughphysical examination and their medical history was documented.Well-being, ketone bodies, mild symptoms and any changes in major health complaintswere self-reported under supervision. The results were daily noted in a questionnaire (S4, S5,S6 Text). A total of 1311 subjects out of the 1422 returned the completed questionnaire.Weight, abdominal circumference, blood pressure and pulse. Clinical data were collected by the physicians. Trained nurses documented every morning according to a standardized protocol the body weight of the participants wearing standard clothing (Seca 704, Seca,PLOS ONE https://doi.org/10.1371/journal.pone.0209353 January 2, 20194 / 23

Safety, health and well-being during periodic fastingHamburg, Germany). Blood pressure and pulse were measured after a pause, once at the nondominant arm in sitting position (upper arm blood pressure monitor, boso Carat professional,BOSCH SOHN GmbH u. Co. KG, Jungingen, Germany). Height was assessed with seca 285(Seca, Hamburg, Germany) and abdominal circumference was determined with a measuringtape mid-way between the lowest rib and the iliac crest (openmindz GmbH, Heidelberg,Germany).Well-being. To evaluate well-being, the participants self-reported daily their physical(PWB) and emotional well-being (EWB) on numeric rating scales from 0 (very bad) to 10(excellent), under nurses’ supervision. The aim was to document the tolerability of the fastingprogram.In a pre-study sample, we evaluated the acceptance of validated questionnaires to assesswell-being within the patient population of the BWC, but found that they were regarded as tootime-consuming in comparison with the numeric rating scales. To avoid drop-out and missingdata, we therefore decided to use numeric rating scales.Ketone bodies. The subjects self-measured the semi-quantitative concentration of ketonebodies in the first morning urine using Ketostix (Bayer AG, Leverkusen, Germany), whichreacts according to the concentration of acetoacetic acid.Mild symptoms and adverse effects. The Buchinger periodic fasting program was continuously monitored for safety and supervised by the medical staff: mild symptoms werereported daily by means of a multiple choice questionnaire, completed by the subjects underthe supervision of nurses. This questionnaire listed the 19 most frequent mild symptoms thatare observed in BWC and mentioned in the guidelines of the Buchinger fasting therapy [31].We considered a mild symptom as being relevant when it was mentioned at least 3 times. Inaddition to the listed symptoms, the medical staff reported further mild symptoms that we categorized as “observed symptoms”. Furthermore, occasional adverse effects (AE) were documented by the physicians.Major health complaint. A self-evaluation of health status was undertaken at the end ofthe stay: the subjects were asked to self-rate any changes in their major health complaint (incases in which they indicated one at the begin of the fast) during the fasting intervention on avisual numeric scale from 0 (much worse) to 7 (much better).Blood analysis. A blood analysis was taken according to international methods (seebelow): (lipid parameters: total cholesterol [TC], triglycerides [TG], high-density lipoprotein[HDL-C], low-density cholesterol [LDL], LDL-C/HDL-C ratio [LDL/HDL-ratio]; glycaemia:blood glucose and glycated haemoglobin [HbA1c]; blood count: leukocytes, erythrocytes, haemoglobin, haematocrit, mean cell volume [MCV], mean corpuscular haemoglobin [MCH],mean corpuscular haemoglobin concentration [MCHC], thrombocytes; coagulation: international normalized ratio [INR], Quick, partial thromboplastin time [PTT]; liver function:serum glutamic oxaloacetic transaminase [GOT], serum glutamate pyruvate transaminase[GPT], serum gamma-glutamyl transferase [GGT], alkaline phosphatase [AP]; inflammatorybiomarkers: C-reactive protein [CRP], erythrocyte sedimentation rate [ESR] after 1 and 2hours; renal function: uric acid, urea and creatinine and electrolytes: sodium [Na], potassium[K], calcium [Ca], magnesium [Mg]).Laboratory examinationsBlood samples were collected twice, namely before the start of the fasting (thereafter referredas baseline values) and at the end of the fasting period. They were collected by trained medical-technical assistants between 7.30 and 9.30 am and drawn into EDTA (S-Monovette 2.7 mlK3 EDTA), citrate (S-Monovette 3 ml 9NC, Citrate 3.2% [1:10]) and blood sedimentationPLOS ONE https://doi.org/10.1371/journal.pone.0209353 January 2, 20195 / 23

Safety, health and well-being during periodic fastingtubes (S-Sedivette 3.5 ml 4NC, ESR/Citrate Buffer [1:5]), that were shaken gently after filling.Additionally, serum tubes including serum gel with clotting activator (S-Monovette 9 mlZ-Gel) were used and stored upright for 30 min until coagulation, with subsequent centrifugation at 3920 g (5000 rpm) for 10 min at room temperature. All tubes were manufactured bySarsteadt AG & Co. (Nürnbrecht, Germany).The ESR was assessed within a period of 4 hours after blood collection and determinedafter 1 and 2 hours of blood sedimentation. All further analyses were performed at MVZ LaborRavensburg, according to the manufacturer’s instruction, in a fully- automated laboratory.Blood cell count (leukocytes, erythrocytes, haemoglobin, MCV, MCH, MCHC, thrombocytes)was measured using the blood analyser Sysmex XN-9000 (Sysmex Europe GmbH, Norderstedt, Germany). Coagulation parameters (INR, Quick, PTT) were assessed on ACL Top (Werfen, Kirchheim, Germany). The liver enzymes (GOT, GPT, GGT, AP), kidney parameters(urea, creatinine, uric acid), lipid parameters (TC, TG, HDL-C, LDL-C, LDL/HDL ratio), electrolytes (Na, K, Ca), glucose and CRP were analysed with ADVIA 2400 (Siemens HealthcareGmbH, Erlangen, Germany). The HbA1c was assessed with TOSOHTM (Bio-Rad LaboratoriesGmbH, München, Germany) and Mg with ICP-MS 7700x series (Agilent, Waldbronn,Germany).Data and statistical analysisParticipants were divided into four groups according to the duration of their fasting period(Fig 1): F5d underwent a fasting period of 5 2 days, with an average of 5.4 (n 695), F10dunderwent a fasting period of 10 2 days, with an average of 8.6 (n 530), F15d underwent afasting period of 15 2 days, with an average of 14.1 (n 196) and F20d underwent a fastingperiod of 20 2 days, with an average of 20.1 (n 37). Between-group differences at baselinewere tested using a one-way ANOVA test followed by Tukey’s post-hoc tests.We tested the effect of fasting, while taking into account the sex and fasting duration groupeffects, by using a multistep parsimonious statistical approach. First, for each outcome theeffect of fasting was assessed by using mixed linear models taking repeated measurementsamong subjects into account, with fasting intervention, fasting duration group, sex, fastingduration group-by-fasting-intervention, fasting intervention-by-sex, sex-by-fasting durationgroup and baseline values of the outcome (pre-fasting) as fixed effects. For each outcome, thecovariance structures was selected among three (compound symmetry (CS), autoregressive(AR(1)) and variance components (VC)) using the Bayesian information criteria (BIC). In alast step the interaction effects that were not significant, were removed from the model toobtain a more parsimonious model. To simplify the presentation of the results, sex differencesare presented in figures only when the fasting-intervention-by-sex effect was significant. Totake into account the multiple tests performed on this dataset, significance was set at a conservative level of p 0.01.Data are shown as mean standard error of the mean (SEM), if not indicated otherwise. Statistical analyses were performed with SAS version 9.4 (SAS Institute, Cary, USA). Graphs weregenerated using GraphPad Prism version 6 for Windows (GraphPad Software, La Jolla California USA).ResultsGeneral parametersThe baseline characteristics of the 1422 adult participants are shown in Table 1. Mean age was55.4 0.4 with 59.1% women and 40.9% men. In total 63.4% of the subjects were non-obese(BMI 30). Grade I obesity (30 BMI 35) was present in 19.5% and grade II or higherPLOS ONE https://doi.org/10.1371/journal.pone.0209353 January 2, 20196 / 23

Safety, health and well-being during periodic fastingTable 1. Baseline characteristics of the subjects.AllDays (d)F5dF10dF15dF20d5 210 215 220 2Subjects, n (%)1422 (100.0)659 (46.3)530 (37.3)196 (13.8)37 (2.6)Men (%)581 (40.9)278 (42.2)214 (40.4)76 (38.8)13 (35.1)Women (%)841 (59.1)381 (57.8)316 (59.6)120 (61.2)24 (64.9)Age, years55.4 0.454.2 0.5 b,c56.3 0.6 a56.4 0.9 a56.4 2.3Fasting length (days)8.2 0.15.4 0.0 b,c,d8.6 0.0 a,c,d14.1 0.1 a,b,d20.1 0.2 a,b,c91.3 0.6b,c,d79.3 0.8b,c,d28.2 0.227.2 0.2b,c,dBMI 25, n (%)404 (28.4)227 (56.2)133 (32.9)41 (10.1)3 (0.7)25 BMI 30, n (%)497 (35.0)232 (46.7)199 (40.0)61 (12.3)5 (1.0)BMI 30, n (%)425 (29.9)155 (36.5)160 (37.6)84 (19.8)26 (6.1)30.0 0.229.2 0.330.3 0.331.3 0.734.0 1.5BMI 25, n (%)74 (12.7)46 (62.2)18 (24.3)10 (13.5)0 (0.0)25 BMI 30, n (%)231 (39.8)117 (50.6)92 (39.8)20 (8.7)2 (0.9)BMI 30, n (%)230 (39.6)94 (40.9)87 (37.8)40 (17.4)9 (3.9)BMI women, kg/m227.0 0.225.7 0.227.3 0.328.7 0.533.3 1.4BMI 25, n (%)330 (39.2)181 (54.8)115 (34.8)31 (9.4)3 (0.9)25 BMI 30, n (%)266 (31.6)115 (43.2)107 (40.2)41 (15.4)3 (1.1)BMI 30, n (%)195 (23.2)61 (31.3)73 (37.4)44 (22.6)17 (8.7)Waist, cm94.0 0.4Weight, kg82.0 0.52BMI, kg/mBMI men, kg/m294.8 0.7a,c,d82.7 0.9a,c,d28.5 0.3a,c,d98.3 1.2a,b,d106.3 2.8 a,b,c86.6 1.6a,b,d96.7 4.0 a,b,c29.7 0.4a,b,d33.6 1.1 a,b,cSubjects were divided into 4 groups according to the fasting lengths: 5, 10, 15 and 20 2 days. Significant differences between the groups are indicated asa, p 0.05 versus F5db, p 0.05 versus F10dc, p 0.05 versus F15dd, p 0.05 versus F20d. BMI, body mass index. Data are presented as mean 001(BMI 35) in 10.3%. Subjects who chose to fast on average for 20 days (F20d) had the highestbaseline body mass index (BMI), the highest abdominal circumference (waist), and largestweight reduction (-8.6 0.3 kg) (each p 0.001). Men had a higher mean BMI at baseline(Table 1).Weight, abdominal circumference and blood pressureAs expected, weight and BMI showed a significant decrease (fasting intervention: p 0.001) inall 4 groups (S1 Table). The weight loss increased with the fasting period length and variedbetween 3.2 0.0 kg for F5d and 8.6 0.3 kg for F20d (fasting duration group-by-fasting intervention: p 0.001). Abdominal circumference also decreased significantly (fasting intervention: p 0.001). The reduction varied between 4.6 0.1 cm for F5d and 8.8 0.8 cm for F20d(fasting duration group-by-fasting intervention: p 0.001). Weight and abdominal circumference reduction were significantly higher (fasting-intervention-by-sex: each p 0.001) in menin all groups (Fig 2A and 2B), compared with women.Baseline values of systolic blood pressure (SBP) and diastolic blood pressure (DBP) werehigher in the groups fasting longer (Fig 3A and 3B). The mean values for the whole cohortdecreased significantly from 131.6 0.7 to 120.7 0.4 for SBP (fasting intervention: p 0.001)and from 83.7 0.4 to 77.9 0.3 for DBP (fasting intervention: p 0.001). The reduction of SBPand DBP was greater in the groups who fasted longer (fasting duration group-by-fasting intervention: each p 0.001) without gender difference (Fig 3A and 3B), stabilizing for the wholePLOS ONE https://doi.org/10.1371/journal.pone.0209353 January 2, 20197 / 23

Safety, health and well-being during periodic fastingFig 2. Changes in weight (A) and abdominal circumference (B) according to the length of fast and 3.g002cohort around 120/78 mm Hg (S1 Table). We did not observe significant changes in heart rateduring fasting in the whole group (S1 Table).Well-beingBaseline values of emotional well-being (EWB) and physical well-being (PWB) were lower inthe groups that fasted longer. This suggests that subjects choosing longer fasting periods hadlower emotional and physical self-ratings at baseline than the ones who selected shorter periods of fast. EWB as well as PWB were both significantly enhanced in the course of the fast(fasting intervention: each p 0.001) (S1 Table). There is no difference between genders forthose parameters (Fig 4A and 4B). All groups reached similar increased values of well-being atthe end of their stay.Ketone bodiesAcetoacetic acid, reflecting ketosis, increased significantly from baseline to the end of fast (fasting intervention: p 0.001), suggesting a plateau value reached after 5 days. Men had higherscores of acetoacetic acid than women (fasting intervention-by-sex: p 0.001) (S1 Table).Mild symptoms and adverse effectsThe safety of the Buchinger fasting program was assessed by collecting daily all self-reportedand observed mild symptoms (Table 2). Of the 1311 participants who returned the filled questionnaire, 0.35% reported muscular cramp, which was the least frequent mild symptom, and14.94% sleep disturbances, which was the most frequent mild symptom. As shown in S1 Fig.PLOS ONE https://doi.org/10.1371/journal.pone.0209353 January 2, 20198 / 23

Safety, health and well-being during periodic fastingFig 3. Changes in systolic (A) and diastolic blood pressure (B) according to the fasting 3.g003the incidence of mild symptoms like muscle pain, sleep disturbances, headaches, and hungeroccurred mainly in the first days of the fast.No fatalities or permanent adverse effects were observed. Two subjects had to be admittedto hospital. A 75-year old man with known coronary artery disease had on the 9th fasting day anon-ST segment elevation myocardial infarction and received uncomplicated percutaneouscoronary intervention. After 3 days in the hospital he returned to BWC. The second case was a67-year old woman who had a one-day hospitalisation because of vomiting with dizziness anddiarrhoea on the 4th fasting day. After returning to BWC she received an 800 kcal/d diet. Theother AE were transitory and did not lead to an interruption of the fasting therapy. AE(Table 2) such as cardiac arrhythmia were low-grade, transitory and could be treated uncomplicatedly without stopping the fasting. The same applied to transitory hypoglycaemia. We alsoobserved one case of gout attack in a patient treated previous to the fasting with allopurinol forhyperuricemia and frequent gout attacks. He was able to be symptomatically treated and wenton fasting.Major health complaintTo document the effects of the Buchinger fasting program on their health we asked the participants to self-report if they had a major health complaint before the fasting and how this condition had been influenced by the fast. A group of 404 subjects out of the 1311 who returned theself-report (S2 Fig) mentioned having a major health complaint previous to the fasting. Theywere asked to evaluate the changes after the fasting. In 84.4% of the 404 subjects the majorhealth complaint had much improved, 8.7% reported that it remained unchanged and 6.9%reported a worsening.PLOS ONE https://doi.org/10.1371/journal.pone.0209353 January 2, 20199 / 23

Safety, health and well-being during periodic fastingFig 4. Changes in emotional (A) and physical well-being (B) during fasting. Self-recorded data of a 0–10 numeric rating scale for a total of 1074volunteers are .g004Blood lipids and glycaemiaWe assessed the impact of the Buchinger fasting program on metabolism by analysing severalblood parameters (S2 Table).The lipid values are indicated in S2 Table and Fig 5. At baseline, TG values of women werelower than the values of men. Fasting reduced TG levels by 0.44 mmol/L on av

Universita t Berlin, Humboldt-Universita t zu Berlin, and Berlin Institute of Health, Berlin, Germany, 8 Department of Internal and Integrative Medicine, Immanuel Krankenhaus Berlin, Berlin, Germany These authors contributed equally