1.       Introduction

Obesity is currently considered a global pandemic; 2.8 million people die each year as a result of being overweight or obese as declared by the World Health Organization (WHO) [1]. In the UK it affects 25% of the population and the NHS has not succeeded at developing coherent policies that address obesity as a major cause of health and social care expenditure [2]. Thus, it is of great importance for the scientific community to identify and investigate effective nutritional interventions to fight obesity. Weight gain is considered a subsequent cause of excessive calorie intake [3], and Calorie Restriction (CR) defined as a reduction in energy intake without malnutrition [4,5], has been proven as an effective strategy to reduce adiposity in humans [6,7,8,9].

However, CR diets have traditionally shown notoriously low long-term success rates [10] and high attrition rates within the first weeks [11]; it has been estimated that only 20% of obese individuals are successful at long-term body fat reduction when defined as losing at least 10% of initial body weight and maintaining the loss for at least a year [12]. Therefore, there has been an increased interest in developing more manageable long-term CR interventions as it is unlikely that constant CR will be widely adopted, mainly due to the difficulty in maintaining long-term low-calorie intake in modern society [3]. Intermittent Fasting (IF), a dietary approach where the frequency of food consumption is altered instead of limiting overall calorie intake has been recently proposed as an alternative to the classic model of CR [13,14].

2.       Continuous Calorie Restriction

CR has been studied in animal models for almost a century, and even though the majority of the studies were aimed to investigate delays in aging and extensions in life span, consistent reductions in bodyweight are always observed: Osborne et al. found significant weight loss among other improved biomarkers of health in rats in 1917 [15], similarly, McCay et al. concluded in 1935 that controlled CR without causing malnutrition in rats compared with rats fed ad libitum effectively reduces body weight and also improves various other health markers [5]. Accordingly, subsequent studies have confirmed similar results attributed to CR in rodents [16,17,18] and also in rhesus monkeys [19].

CR in humans promotes weight loss and changes in body composition, and it has been associated with improvements of different markers for cardiovascular and metabolic health in both overweight [20,22] and non-overweight subjects [22,23]. As dietary intervention, CR is seen as a reduction of usually 20-40% of daily calorie requirements [18] while maintaining adequate nutrition over a certain period of time [24], which has been traditionally applied in humans in the form of either a Low-Calorie Diet (LCD) providing around 1000 to 1500 kcal/day [25,26,27,28] or a Very-Low- Calorie Diet (VLCD) providing <800 kcal/day [29,30, 31,32]. While at short-term the VLCDs appear to be superior for initiating changes in body composition, the long-term effects on sustained weight loss seem to be very similar for both CR interventions [29]. In fact, a meta-analysis of randomized trials comparing long-term efficacy of LCDs vs VLCDs conducted by Tsai and Wadden concluded that even though VLCDs lead to greater results at first, they do not produce greater long-term weight losses than LCDs [25].

The negative side effects reported on long-term CR for humans are similar to those observed in animals: perpetual hunger, reduced body temperature leading to cold intolerance, and diminished libido [33] while rare adverse effects reported on long-term VLCDs are mild postural light-headedness, fatigue, decreased bowel movements and constipation, electrolyte disturbances, dry skin and hair loss [34,35]. Another important drawback observed in long-term studies seems to be the adaptive decrease in Total Energy Expenditure (TEE) linked to prolonged periods of CR seen in Biosphere experiments [36,37,38].

3.       Intermittent Fasting

Even though some researchers support the feasibility of long-term CR [39], adherence to the recommended CR diets remains an issue for the majority of the population in the long term [40]. Another major drawback of CR is that, according to Johnstone, subjects following both LCD and VLCD diets tend to regain all of the weight lost one year after their initiation of the diet [41]. It has been hypothesized that the reported beneficial effects from CR on body composition and other health biomarkers can be mimicked by alternating periods of short term fasting with periods of refeeding, without deliberately altering the total caloric intake [42,43,44].

IF programs for weight loss in obese and non-obese subjects have been recently classified as Whole Day Fasting (WDF), Daily IF (DIF) and Alternate Day Fasting (ADF), each form of IF utilises different periods of feeding and fasting [45] (Table 1). They are currently gaining popularity in the lay press and among research scientists [46], common models are the 5:2 diet (an ADF protocol of 500 kcal/day two days per week) and the 18:8 diet (a DIF protocol of no calories during 16 hr and an eight-hr feeding window over a 24-hr period) [46]. DIF protocols like the 18:8 diet have been supported by some research such as the crossover study conducted by Kahleova et al. which found that daily small feeding windows (two meals per day skipping dinner) produced significantly greater weight reduction than grazing (six smaller meals per day) [47] and other studies that found similar results on weight loss [13,48,49] however, the 5:2 model seems to be more popular at the moment and it is more utilised in research [50,51,52,53].

IF has been widely studied in animal models, most often in the form of Every-Other-Day feeding (EOD), where they have no food for 24 hr and Ad Libitum (AL) access to food during the next 24 hr [54]. In many rodent species, mice managed to compensate for the calorie deficit created during the fasting days by increasing their calorie intake on the AL feeding days [55], over a two-day period, they kept their total caloric intake at the same level as in mice fed an ad libitum diet. In human trials seeking to replicate the effects reported in rodent studies, IF in the form of ADF has consistently shown significant reductions of body weight in only three weeks [56,57]. In studies where participants underwent modified versions of ADF similar to the 5:2 models, most trials found weight reductions in all of the participants [58], although some researchers found significant weight decreases in obese subjects [59,60], while bodyweight was maintained in lean individuals [61,62].

4.       Common Misconceptions and Potential Adverse Effects from IF

4.1.  Decreased Metabolic Rate

It is widely believed that IF leads to decreases in human metabolism and that multiple small meals cause the opposite effect increasing the overall energy expenditure due to the Thermic Effect of Food (TEF) [63], but the research on metabolic rate is remarkably conclusive: TEF is dependent on the total caloric intake and changes with macronutrient variability and not meal frequency [64,65, 66,67]. Webber and Macdonald studied the metabolic effects of fasting for 12, 36 and 72 h in 29 participants and found no changes in their metabolic rate [68]. Heilbronn et al. demonstrated that ADF does not affect resting metabolic rates in healthy males and females [56]. Gjedsted et al. examined the effects of 72 h fast on 10 lean men, at the end of trial the subject’s energy expenditure was found to be unchanged from the initial measurements [69]. Similar data has been found regarding continuous CR diets; no decreases in metabolic rates were found after a 12-week trial that examined the effects of a VLCD of 800 kcal/day on subjects undergoing resistance training [70], the same results were found in another study where 30 female subjects underwent a 72h severe CR intervention [71].

4.2.  Low Blood Glucose Levels

It is commonly believed that IF can cause pathologically low levels of blood glucose in non-diabetic subjects, however, research shows that in healthy subjects short-term fasting periods of up to 24 h do not lead to hypoglycaemia [72]. In fact, no data has been found where subjects undergoing IF interventions reported glucose levels below 3.6 mmol/L. Alken et al. studied individuals that reported a history of hypoglycaemic episodes and compared them to subjects that have never experienced any form of hypoglycaemia. Both groups completed a 24 h fast while their glucose levels were monitored and none of the participants showed any periods of hypoglycaemia, although the group that had a history of hypoglycaemia reported periods of ‘feeling hypoglycaemic’ even though their blood glucose was at normal levels [73], the study concluded that despite reported ‘hypoglycaemic sensations’, in the absence of metabolic disease, the ability to maintain blood glucose levels within the normal range does not seem to be affected by short periods of fasting. Award et al. concluded that blood glucose levels are kept in the normal range after a 24 h fast mainly because the liver can store enough glycogen; in fact, they found that fasting for 24 h only decreased 57% of the liver glycogen stores in healthy individuals that were not engaged in vigorous exercise [74].

4.3.  Increased Hunger

It is widely accepted that eating smaller meals frequently as opposed to restricting feeding frequency helps reduce hunger perception in humans [75,76,77]. Such assumption has linked IF interventions with a possible increment of feelings of hunger, which is indeed a major limiting factor for adherence and compliance in most dietary interventions. However, perceived hunger appears to be highly subjective [78,79] as food ingestion seems to be modulated by both peripheral and central signals [80], suggesting that feelings of hunger are greatly dependent on the individual’s preferred feeding pattern [81]. Studies on starvation have shown decreased feelings of hunger: Duncan conducted a 14-day fast experiment in obese patients who were only allowed to consume water and non-caloric beverages. The intervention caused great reductions of body weight but did not cause increased perceived hunger sensations, demonstrating that prolonged fasts might have a hunger suppressing effect [82], although the results might not transfer to the short duration fasting periods of IF protocols. Heilbronn et al. studied the perception of hunger and fullness in 18 males and females that followed an ADF IF protocol during 22 days and found that subjects reported increased feelings hunger on fasting days [56]. However, Johnson et conducted a similar study where 10 obese subjects underwent an eight-week ADF intervention and hunger perception did not increase significantly from baseline during the trial period [60].

To date, research on the effects of IF on perceived hunger is limited, and even though there seems to be a greater amount of data on hunger and feeding frequency, it is not conclusive and studies frequently find conflictive results. Speechly et al. studied different feeding frequencies and the relationship between hunger and subsequent food intake in obese men. Participants were fed 33% of

their daily calorie requirement in either one single meal or five meals before being allowed to eat ad libitum. The single meal group consumed 27% more calories when given the ad libitum meal [83]. The same setup has been used in lean individuals finding similar results, researchers concluded that when the dietary load was spread into equal amounts and consumed evenly through the day, appetite control was enhanced [84].

Contrary to the common believe, some studies suggest that more frequent meals throughout the day lead to increased hunger levels: Smeets and Westerterp-Plantenga conducted a randomised crossover study where 14 females were given either two or three meals per day in a respiration chamber for measurements of energy expenditure and substrate oxidation. They concluded that in healthy, non-obese women, decreasing the feeding frequency sustains satiety [85]. Similarly, Munsters and Saris investigated the effects of meal frequency in 12 healthy males that randomly received two isoenergetic diets with either three or 12 meals a day. The low-frequency diet increased satiety and reduced hunger ratings compared to the high-frequency one [86]. Ohkawara et al. found similar results in a randomized cross-over study that compared the effects of consuming three vs six meals in 15 lean male and female subjects; They found no difference in fullness, but hunger and “desire to eat” were greater during six compared to three meals [87]. Very recently, Perrigue et al. conducted a randomized crossover intervention trial in 12 healthy males and females to examine the effects of high vs low feeding frequency on self-reported appetite and found the same results, concluding that frequent meals do not help to decrease overall appetite when compared to restricted feeding frequency [88].

4.4.  Increased Stress

Fasting research in animal models have shown that ADF forms of fasting can increase adrenocorticotropic hormone and cortisol levels in rodents [89,90], which has led to the assumption that IF produces the same effects in humans [91]. However, research shows that the controlled stress response from IF interventions in humans seems to be different from the one by uncontrolled physiological and psychological stress seen in rat studies, and the possible increased stress in humans might be a necessary factor for initiating molecular resistance for larger stressors that can promote beneficial effects [16]. In fact, short periods of increased cortisol secretion such as the ones seen during some IF interventions can enhance fatty acid oxidation, while more prolonged cortisol increases can have negative effects such as causing vulnerability to immunosuppression, and to autoimmune related and metabolic disorders [92]. Most research on fasting show little or no changes in cortisol levels in response to short periods of fasting: Soeters et al. conducted a crossover study where lean healthy subjects underwent two weeks of an ADF fasting protocol of 36 hours fasts and found no negative effects on cortisol levels [62]. Similar interventions have found the same results after fasting periods of up to 24 hours [93] and even after 72 hours of total calorie deprivation [94], although 72 hours of fasting has been shown to increase cortisol levels in very lean females [95]. Research shows that sustained periods of fasting can indeed lead to significant increments of cortisol levels in humans; Bergendahl et al. found that five days of fasting caused a 1.8-fold increase in the 24-hour endogenous cortisol production rate in non-obese healthy [96], but the duration of the fasted stage of normal IF protocols do not exceed 24 hours.

4.5.  Loss of Lean Mass

It is commonly believed that it is important to have a steady stream of amino acids available to avoid muscle catabolism, and it has been hypothesised that IF can lead to depleted liver glycogen stores in humans, increasing proteolysis and flux of amino acids from skeletal muscle for hepatic de novo gluconeogenesis [42]. However, research does not seem to suggest that short periods of fasting of up to 24 hours can deplete hepatic glycogen stores in healthy individuals [74], and caloric deprivation of up to 40 hours does not appear to stimulate a significant catabolic effect or amino-acid breakdown [97]. Fasting increases ketone body concentrations [60] which have been shown to have an anti-catabolic effect and also provide a non-glucose energy substrate for the body decreasing the need for protein-derived substrates for gluconeogenetic conversion [98,99]. Soeters et al. conducted a crossover study where eight healthy subjects underwent a two weeks ADF IF protocol and a two week of a standard diet and they found no significant loss of lean mass [62]. Gjedsted et al. also suggest that fasting for up to 72 hours does not correlate with an increased breakdown in the muscle and it does not slow down muscle protein synthesis in healthy individuals [94].

It is important to mention that the majority of the literature on the preservation of muscle mass during calorie deprivation or reduction involve some form of resistance or anaerobic exercise: Bryner et al. studied male and female subjects consuming 800kcal/day during a 12-week intervention that participated in resistance exercise three days a week. Researchers found that all participants were able to maintain their fat free mass [70]. Another study conducted in 1999 studied obese males over a 16-week period controlling their caloric intake by reducing their daily intake by 1000 calories per day. Researchers found that whilst the participants attended a weight training programme three times a week, they were able to lose over 20 pounds of body fat and still maintain all muscle mass [100]. Janssen et al. carried out a similar study into 38 obese women who had reduced their calorie intake for 16 weeks and participated in weight training three times a week. The findings also showed that the participants were able to maintain their muscle mass [101]. Cchomentowski et al. looked into 29 males and females between the age of 60 and 75 who had dieted for 4 months. The results underline that the group that did not participate in exercise had over 4% decrease in lean body mass, whereas those who had participated in exercising had no significant decrease in lean mass.

5.       Rationale for the Present Study

From the current body of research, IF appears to produce similar effects to continuous CR, being able to reduce body weight and adipose tissue in normal-weight, overweight, and obese individuals [45], although it has been speculated that the calorie deficit from the fasting periods could precede weight gain due to over-eating during the ad libitum feeding days, which is something that has been seen in some rodent species [55]. Most animal research shows a number of positive effects IF on weight and fat distribution [102], however, scientific evidence for the benefits of IF in humans is often extrapolated from the mentioned animal trials, based on observational data, or derived from experimental studies with inadequate sample sizes [44,103]. To date, there is a scarce number of studies in healthy non-obese individuals comparing IF with CR and there is currently no data comparing IF with CR in normal-weight subjects [104]. Further studies comparing IF vs CR for weight loss and body composition changes in normal-weight populations will likely add valuable data to the available knowledge, as more research is needed to establish the effectiveness of one method over the other.

5.1.  Aim

To compare the short term (5 weeks) effects on weight and body composition of the popular IF protocol 5:2 against a conventional CR diet in healthy non-obese individuals.

5.2.  Objectives

The objectives of this study are to: (i) compare the effectiveness of IF with continuous CR for weight loss; (ii) compare the effectiveness of IF with continuous CR for changes in body composition.

5.3.  Hypothesis

It has been hypothesised that IF and CR protocols will both lead to short-term weight loss and body composition changes in healthy non-obese individuals. However, subjects on the IF diet might compensate for the calorie deficit created during the fasting periods by increasing their calorie intake on the ad libitum feeding days, as seen in animal models [39]. This would make the CR protocol more effective for weight loss and body composition changes.

5.4.  Methods

5.4.1.        Subjects

Fourteen physically active, non-obese (BMI < 29.9 kg/m²) healthy males (n=4) and females (n=10) aged 20-30 years and primarily Caucasian that have been recruited at the Peckham Pulse Healthy Living Centre, 10 Melon Rd, London SE15 5QN. Participants’ measurements have been taken at baseline and upon completion of the trial at Haymerle School’s Gym, Haymerle Road, Peckham London SE15 6SY.

Participants have been required to be non-smokers, not dieting before the trial or losing weight and have regular menstrual cycles; they were required not to consume high intakes of alcohol (>28 units per week) and not be diagnosed with diabetes, cardiovascular disease or cancer.

Approval was obtained from the St Mary’s University, Twickenham ethics sub-committee. Prior to the study initiation, each participant was provided with a Consent Form and Information Sheet (Appendices 1 and 2).

·         Appendix 1 - Participant Information Sheet

Participant Information Sheet

You Will be Given a Copy of this Form to Keep Together with a Copy Of your Consent Form

·         Appendix 2 - Participant Consent Form

5.4.2.         Design

Randomized comparison of the participants divided in two groups: (i) IF group - Periodised severe energy restriction (500 kcal/day for 2 days/week) and (ii) CR group - Continuous caloric restriction (1200 kcal/day for 7 days/week) observed over a period of five weeks. The IF protocol has been based on previous studies on the common version of 5:2 IF diet which generally involve severe caloric restriction (75 - 90% restriction of energy needs or an intake of 200 - 500 kcal/day) on non-consecutive 2 days per week [53,]. The CR protocol has been based on a recent systematic review and meta-analysis of randomized controlled trials that classified LCDs as diets where total calorie intake is <1200 kcal/day [105].

Since heavier subjects were considered likely to lose weight and adipose tissue faster than leaner individuals [106], it was considered that starting BMI may confound the rate of weight loss during the trial. A Randomized Block Design has been used where BMI is the blocking variable; Block one clusters participants with lower BMI scores (BMI = 18-22 kg/²) and block two clusters higher scores (BMI = 23-30 kg/m²). For equal allocation, odd participant numbers have been assigned to the CR dieting group and even participant numbers have been assigned to the IF dieting group.

The length of the intervention has been based on Barnosky’s review of the scientific literature on IF which concludes that significant fat loss can be seen from the third week [50], CR interventions also tend to be studied during periods of around five weeks [28,29,53] as the weight reduction normally appears to be significant after the fourth week.

Weight and body composition have been assessed at baseline and after the fifth week. The anthropometric assessment for body composition consisted in the 4-site skinfold Jackson and Pollock equations where the skinfold sites are abdominal, triceps, thigh and suprailiac to calculate the % of sub-cutaneous body fat [107] (Appendix 3), as they are widely used in research and highly effective in non-obese populations [108]. Weight have been assessed with a Salter digital scale and skinfolds have been taken using a Harpenden (HRP) caliper [109,110].

·         Appendix 3 - Jackson and Pollock anthropometric equations for body composition

During the initial session of anthropometric measurements both groups of participants received detailed information regarding the dietary intervention and were able to ask questions to the main investigator. Both groups received examples of weekly meal plans (Appendices 4 and 5).

·         Appendix 4 - IF group 5:2 protocol

IF group 5:2 protocol

The 5:2 Intermittent Fasting protocol requires that you consume only 500 calories a day during two non-consecutive days a week and eat unconstrainedly the other five days of the week. It is very important that you leave at least one or two days of ad libitum eating between your weekly fasting days.

Remember to drink plenty of water throughout your fasting days. You can drink tea and coffee during your fasts, but you cannot add sugar, milk or cream, which contain calories. Use artificial sweeteners for flavour if you like. Avoid the coffee drinks in coffee shops if you are not sure whether they contain some type of syrup, milk product or sugary add-in.

·         Appendix 5 - CR group protocol. Weekly meal plan - Approximate daily intake: 1200kcal

5.4.3.         Analysis

Data from weight and body composition of the subjects have been analysed using the Statistical Package for Social Sciences (IBM 2015). Descriptive data and weight and body composition data at baseline and upon completion of the trial are presented as mean and standard deviation. Dependent (paired sample) t-tests were carried out to assess differences in weight and body composition before and after the intervention in both groups. Normality was assessed by Shapiro- Wilk’s test and independent t-tests were carried out to examine weight loss and sub-cutaneous body fat reduction differences between each diet group (Appendix 6). The level of significance for all tests has been set at p < 0.05. Dependant variables: Weight (ratio), % body fat (ratio); Independent variables: Diet (nominal), gender (nominal), age (ratio).

·         Appendix 6 - Raw SPSS outputs

Descriptive Statistics

5.4.4.         Results

Descriptive characteristics of subjects at baseline are reported in (Table 2). All participants were of comparable age, height, BMI and were mainly Caucasian (Appendix 6).

Table 3 shows the descriptive statistical analysis of the weight and body composition data obtained at baseline and upon completion of the intervention (Appendix 6).

A dependent (paired sample) t-test was carried out to assess whether there is a significant difference between weight before and after the intervention in both dieting groups. The t-test revealed that the IF protocol had produced a statistically significant change in bodyweight (t = 4.69, p < 0.05) but the CR protocol had not produced a statically significant change in bodyweight (t = 0.34, p > 0.05) (Table 4) (Appendix 6) (Figure 1).

A dependent (paired sample) t-test was carried out to assess whether there is a significant difference between % of body fat before and after the intervention in both dieting groups. The t-test revealed that the IF protocol had produced a statistically significant change in % of body fat (t = 3.7, p < 0.05), the CR protocol had not produced a statically significant change in % of body fat (t = 1.02, p > 0.05) (Table 5) (Appendix 6) (Figure 2).

Weight loss differences within each diet group have been calculated (initial weight minus weight after intervention); Sub-cutaneous body fat reduction differences within each diet group have been calculated (initial sub-cutaneous body fat minus sub-cutaneous body fat after intervention). Weight loss and sub-cutaneous body fat reduction differences in the IF group have been compared against weight loss and sub-cutaneous body fat reduction in the CR group. The differences were normally distributed within each diet group, as assessed by Shapiro-Wilk’s test (p > 0.05) (Appendix 6).

An independent t-test was carried out to determine whether the weight loss and sub-cutaneous body fat reduction differences between each diet group were statistically significant. Levene’s Test for Equality of Variances revealed that equal variance could be assumed for weight loss (F = 0.80, p > 0.05) and body fat reduction (F = 0.26, p > 0.05) (Appendix 6). The independent t-test revealed that the IF protocol had produced statistically significant greater weight loss (0.8 ± 0.4 kg) and body fat reduction (1 ± 0.6 %) compared to the CR protocol (0.1 ± 0.6 kg for weight loss) (t = -2.4, p > 0.05) and (0.3 ± 0.7 % body fat reduction) (t = -1.7, p > 0.05).

6.       Discussion

6.1.  Main Findings

The aim of the study was to determine changes in weight and body composition between commons IF and CR protocols in healthy non-obese individuals over a five-week intervention. Even though it was initially hypothesised that the IF group could have compensated for the calorie deficit created during the fasting periods by increasing their calorie intake on the ad libitum feeding days, the IF dietary approach has achieved significant weight loss and body composition improvements, whereas the CR approach has not produced any significant changes in weight or body composition.

7.       Comparison with Previous Literature

7.1.  IF Results

The significant weight loss and body composition improvements achieved by the IF protocol appear to agree with most of the literature on IF. Previous studies have established that IF methods are known to be as effective as daily CR for weight reduction, body composition and weight control in humans [58]. It has been proven that as long as subjects undergoing IF interventions do not compensate by over-eating during the non-fasting periods, IF will always lead to reduced calorie intake [41, 50]. In line with our results for the IF group, most IF studies have found significant improvements on weight and body fat [57, 59,60,61,111,112,113].

Our IF results for weight loss (0.8 ± 0.4) kg are not as strong as those found in similar trials of comparable duration: Johnson et al. conducted a 4-week ADF intervention and achieved 4kg of total weight loss [60], Eshghinia’s first study found 4.7 ± 2.5kg of total weight loss after a similar 4 week ADF protocol [114] and achieved 6 ± 1.2kg after a 6-week ADF second study [113,118], Varady et al. achieved 5.6 ± 1kg with a similar ADF design, although the trial lasted 10 weeks [59]. According to these effects on body weight, our IF results for body fat reduction (1 ± 0.6) % are not as strong as those found in the above trials of comparable duration which reported changes in body composition: Eshghinia and Mohammadzadeh’s study found a body fat reduction of 2.84 ± 0.2% and Varady’s trial found a body fat declined of 2.9 ± 1% [59].

However, the mentioned trials utilised more severe forms of IF and often times participants were overweight or even obese, which may explain the weight loss differences compared to our study.

When examining other IF studies where researchers applied dissimilar fasting protocols to the present study, positive improvements are always found: Barnosky et al. conducted an IF vs CR for type 2 diabetes prevention study where they observed weight reductions of 0.25kg of body weight per week and also found reductions of waist circumference on an IF regime with a 75% energy restriction during the 1-3 fasting days per week [50]. Heilbronn et al. conducted another interesting study where participants lost an average of 2.1kg total bodyweight after a 22-day ADF intervention despite given instructions to eat double their typical day’s intake every feeding day [56]. Studies also looking at the effects of IF on different health markers such as decreased blood pressure, resting heart rates, glucose metabolism and cardiovascular health have consistently shown improvements after implementing IF protocols [4, 61,62]. More aggressive IF protocols are seen in the literature, such as the one utilised by Johnstone et al. where lean subjects underwent a 36 h fast that resulted in a negative energy balance of approximately 12 MJ; researchers found that participants lost an average of 1.3kg of total body weight [115], which matches the exact same amount of weight loss (1.3kg) found after a single 24 h fast in one of the early clinical studies on IF [116], although most of it was thought to be intramuscular glycogen and water. Again, most of these studies have examined overweight or obese participants. It is also important to mention that no IF protocols for weight loss recommending fasting periods over 24 h outside of a clinical setting have been found.

7.2.  CR Results

Our CR results for both weight loss (0.1 ± 0.6) kg and body fat reduction (0.3 ± 0.7) % are statistically non-significant, data that do not seem to agree with the vast majority of the scientific literature. While research on CR in humans is still at an early stage [33], the effects on body weight and changes in body composition seem to be consistent in most studies: from early trials in humans concluding that CR is an effective strategy towards weight loss [117,118,119,120,121,122] to the current research that confirms a direct correlation between CR and body composition [123,124,125,126].

The positive correlation between CR and weight and body changes has been shown in short term trials of a similar duration to the present study where participants have experienced significant improvements: Stamets et al. examined the effects of CR intervention in obese women and after only 4 weeks of hypocaloric dieting participants experienced a weight reduction of 4.1 ± 1.7 kg [127], Norrelund et al. also conducted a 4 weeks trial on obese individuals where they examined the effects of a VLCD diet on protein breakdown [128], upon completion of the study participants reduced total body weight by 5% and body fat by 10%. Trials of longer duration have shown greater improvements for both weight and body composition: Haugaard et al. and Luscombe et al. utilised similar CR protocols during 8 weeks in obese and overweight participants, the first trial found an 8% body weight reduction and a 14% body loss [129], the second found the same percentage of weight loss and a 20% of body fat reduction [130]. The same trend is seen in long-term trials where the correlation between CR and weight and body composition changes has also been well established, such as the coordinated multicentre study CALERIE (Comprehensive Assessment of Long Term Effects of Reducing Caloric Intake) in which healthy volunteers underwent the CR interventions for two years to test how practical and safe is a 25% CR diet in non-obese subjects, researchers found that participants lost an average of 10% of their body weight in the first year, and maintained the weight over the second year [131,132]. Even though the study did not focus solely on changes in body composition and weight, the results from the data collected are consistent with previous studies related to reductions in body weight and fat [133, 134,135]. Similarly, the Biosphere studies have demonstrated substantial weight loss in subjects after a low- calorie nutrient-dense diet intervention over two years [37,36]. Cross-sectional data on the effects of long-term CR in humans obtained from participants undergoing six years of CR also shows a dramatic reduction in body adiposity and Body Mass Index (BMI) [136,137] as well as improvements in other health markers seen in previous human and animal studies [39].

In previously mentioned studies such as the Biosphere trials researchers were able control participants’ dietary intake in detail, other studies utilised either whole-body calorimeter measurements or a controlled environment for metabolic assessment to accurately study dietary intake data. However, similar trials to the present study where dietary intake assessment is dependent on self-reports or where free-living subjects are expected to estimate caloric intake and food portions tend to face under-reporting and under-estimating issues [138]. Based on our results, and even though participants did not report problems following the diet, we believe subjects undergoing the CR intervention might have experienced adherence problems and could have unintentionally underestimated total food intake.

7.3.  IF vs CR Results

To date, no studies on CR vs IF showing identical results to the present trial have been found, although there has only been limited research directly comparing both dietary protocols. Wing et al. utilised an intermittent VLCD protocol that could be considered IF and compared it to a CR one in a 50-week weight loss trial for obese patients with type II diabetes, they found that subjects following the first protocol lost significantly more weight than did CR subjects during the trial [139]. The authors did not asses body composition changes and even though the first protocol produced better improvements similarly to what we have seen in our study, the CR diet also led to statistically significant weight loss.

Varady’s studies have also examined weight and body composition outcomes as well as Coronary Artery Disease (CAD) risk indicators after different IF and continuous CR interventions in short-term trials of 10 and 12 weeks in obese adults [4, 50,53,59,112]. Researchers have concluded that while IF is a safe and valid strategy to reduce total calorie intake, whether it is superior than continuous CR is still unclear. Seimon et al. published a large systematic review on CR vs IF for body composition changes, weight loss and several other biomarkers. They reviewed 40 human studies on different CR interventions, intermittent energy restriction and IF protocols, 12 of which were a direct comparison of CR vs IF interventions including overweight and obese participants [140], researchers concluded that both dietary protocols appear to induce equivalent weight loss and body composition outcomes, and concluded that neither of them seems to be superior to the other.

Two recent systematic reviews of randomised clinical trials have shown that IF appears to represent a valid option to continuous CR for weight loss, as the achieved weight loss in overweight and obese individuals undergoing IF interventions was comparable to that achieved by traditional CR interventions [140,141]. Davis et al. also concluded that IF represents an effective alternative strategy for health practitioners to promote weight loss in obese and overweight patients. Another recent systematic review and meta-analysis on IF weight and biological markers in long term intervention studies of >6 months duration have conluded that even though IF studies showed weight loss, there was no evidence that it provided specific benefits over continuous CR at least in the little long-term evidence available [126], although researchers concluded that future longer-term trials are required to fully evaluate any lasting benefits that IF might have over traditional CR.

8.       Strengths of the Study

A large proportion of the available trials on both CR and IF does only assess subjects’ body weight to evaluate the effectiveness of the interventions and also tend to measure different biomarkers such as blood glucose or total cholesterol [52, 53, 55,142], however, many of them do not account for body composition changes. The combination of both body composition and body weight has been seen in a limited number of previous CR and IF trials [59,61,111] and it represents an important advantage of the present study since it provides a more precise evaluation of the intervention’s efficacy [56] since research shows that the part of the weight loss produced by prolonged isocaloric dieting is often caused not only by the reduction of adipose tissue but also by amino acid catabolism in muscle tissue [128].

Another advantage of the present study is the fact that we have studied both dietary protocols on physically active, non-obese (BMI < 29.9) [143] healthy subjects, which appears to be a novelty on IF vs CR research. According to Harvie and Howell’s recent summary of evidence, to date, there are currently no data comparing IF with CR in normal-weight subjects [104].

One of the main findings is the difference in efficacy between the two protocols, as we believe it highlights the simplicity of the IF approach against the complexity of traditional CR [44], which seems to be one of the main difficulties with traditional CR [144,145]. Research shows that only 25-30% of individuals that undergo daily CR manage to adhere to it for more than 12 months [146]. In fact, data from independent reviewers and meta-analysis suggest that CR promotes short-term weight loss but the weight reduction has not been proved to be sustainable in the long-term [147].

Contrarily, recent data on IF is showing greater adherence rates for IF protocols: Harvie et al. compared a traditional CR Mediterranean diet against two WDF diets similar to our IF intervention. The IF protocols showed 76% and 74% adherence rates against 39% from the traditional CR diet [148]. In like manner, Wegman et al. studied the practicality and tolerability of IF and found strict adherence to the IF protocols, and all participants found the intervention as tolerable [149]. IF has indeed been described as a plausible weight loss aid [150] as it can improve compliance in human subjects [42,58]. In the US, it has been reported that 14% of adults have already used fasting as a means to control body weight [41], probably due to the fact that IF protocols provide a simple and relatively easy dieting option [151].

9.       Study Limitations

The lack of weight loss and body composition changes found in the CR group could be explained by the nature of the dietary regimes, even though no participants withdrew from the study, the authors speculate that the IF protocol was probably easier to adhere to than the CR one. This in fact is considered one of the main strengths of the IF approach [53, 58, 150,152]. Both groups of participants received detailed information regarding the dietary intervention at baseline and were expected to follow them; however, while the IF protocol mostly requires that participants just avoid consuming any calories during a certain amount of time [14], the CR group had to estimate food portions and calculate calories on a daily basis, and even though they were given a simplified and detailed sample of daily meal plans, the well-established prevalence of underestimation of food intake [153] a free-living population might have resulted in the CR group dieting over their daily calorie target; which seems to be the main limitation of CR as supported by the literature [40,46].

Difficulties in the ability to perceive calories and intake autoregulation have been studied for decades [154] as problems with estimates of food quantity and calories are very commonly found in the literature. The causes of underestimation include inaccuracy in reporting food portions, psychological behaviours, social and cultural factors, memory disturbance and behavioural factors [155]. Avoiding food intake underestimation is in fact considered one of the main challenges that researchers encounter in trials with free-population, and even though food intake underestimation is considered especially extreme in overweight and obese individuals [156,157], it also appears to be prevalent among non- obese populations [158] such as our CR group sample.

10.   Implications and Recommendations for Future Research

Based on the data obtained, the following topics for future research are recommended:

Additional research is needed on IF vs CR for weight loss and body composition changes in normal-weight individuals, since no other studies on normal weight subjects have compared both protocols [104] and obese and overweight studies have shown equal success rates for both.

Since IF has already been established as a valid alternative to continuous CR [44,103, 159], further research is needed to compare different IF versions and to establish the effectiveness of one IF method over the others. To date, no studies comparing different IF protocols against each other for weight loss and body composition changes have been found.

Additional research is also needed to further examine IF vs CR differences in adherence rates, as our study has shown higher compliance for IF in line with recent research [149] and several authors have speculated that IF regimes could improve adherence [58].

More research is need especially regarding long-term IF adherence in free-living populations, since CR has extensively shown poor long-term success [147] and IF might offer a reliable alternative. Further psycho-social data is also required to better examine behavioural factors that can modulate compliance to both diet protocols.

Future IF vs CR studies should always control food intake to avoid intake underestimation. As a preventive measure, we recommend that researchers always include regular food diaries during the trial, although this practise is common in CR studies of similar design [140,152,160,161]. Nevertheless, data is not conclusive on whether or not they can mitigate or reduce the prevalence of underestimation of food intake in free-living populations [138]. If possible, further trials should compare both protocols in settings where researchers are able to provide participants with all meals [40, 128,162], method that guarantees the absence of calorie underreporting and underestimation as researchers control total calorie intake [163]. Alternatively, researchers could utilise more rigorous methods to assess calorie intake to avoid underestimation; for example, in conjunction with food diaries, accuracy could be improved by contrasting reported energy intake with reliable measurement methods such as doubly labeled water measurements or even respiratory-chamber indirect calorimetry systems as they are well established and precise methods for metabolic assessment [164,165,166].

11.   Conclusions

Our data suggest that IF is an effective strategy for weight loss and body composition improvement, as it provides a simplified method for reducing total energy intake that could possibly be easier to adhere than continuous CR. The complexity of daily CR seen in the literature might have influenced the presumed low adherence to the CR protocol in the present study. In a similar fashion, the positive adherence to the IF appears to be in line with recent IF research that shows improved adherence over traditional CR. Further research is required to better explain behavioural and physiological factors, that can enhance or reduce compliance to IF and CR interventions.

IF could potentially be a more practical method for achieving caloric deficit than continuous CR in individuals that tend to underestimate and underreport food intake. Thus, different IF protocols can be offered as an alternative to more traditional approaches of CR for reducing total body weight and optimising body composition.

Figure 1: Mean body weight at baseline and upon completion of the intervention in both groups (error bars represent + 1 SD).

Figure 2: Mean body fat at baseline and upon completion of the intervention in both groups (error bars represent + 1 SD).

1.       Saura J, Isidro F, Heredia JR, Segarra V (2014) Evidencias científicas sobre la eficacia y seguridad de la dieta proteinada: dieta proteinada y ejercicio físico. Andalucia Medicina del Deporte 1: 35-79.

2.       Barth JH, O'kane M (2016) Obesity services: how best to develop a coherent way forward. Clinical endocrinology 21: 175-198.

3.       Fontana L, Klein S (2007) Aging, adiposity, and calorie restriction. Journal of the American Medical Association 297: 986-994.

4.       Varady KA, Hellerstein MK (2007) Alternate-day fasting and chronic disease prevention: a review of human and animal trials. American Journal of Clinical Nutrition 86: 7-13.

5.       Mccay CM, Crowell MF, Maynard LA (1989) The effect of retarded growth upon the length of life span and upon the ultimate body size. Nutrition 5: 155-71.

6.       Heilbronn LK, De Jonge L, Frisard MI, Delany JP, Larson-Meyer DE, et al. (2006) Effect of 6-month calorie restriction on biomarkers of longevity, metabolic adaptation, and oxidative stress in overweight individuals: a randomized controlled trial. The Journal of the American Medical Association 295: 1539-1548.

7.       Larson-Meyer DE, Heilbronn LK, Redman LM, Newcomer BR, Frisard MI, et al. (2006) Effect of calorie restriction with or without exercise on insulin sensitivity, beta-cell function, fat cell size, and ectopic lipid in overweight subjects. Diabetes Care 29: 1337-1344.

8.       Redman LM, Heilbronn LK, Martin CK, Alfonso a, Smith SR, et al. (2007) Effect of calorie restriction with or without exercise on body composition and fat distribution. Journal of Clinical Endocrinology and Metabolism 92: 865-872.

9.       Goldstein DJ (1992) Beneficial health effects of modest weight loss. International Journal of Obesity and Related Metabolic Disorders 16: 397-415.

10.    Grayson BE, Hakala-Finch AP, Kekulawala M, Laub H, Egan AE, et al. (2014) Weight loss by calorie restriction versus bariatric surgery differentially regulates the hypothalamo- pituitary-adrenocortical axis in male rats. Stress 17: 484-493.

11.    Feuerstein M, Papciak A, Shapiro S, Tannenbaum S (1989) The weight loss profile: a biopsychosocial approach to weight loss. International Journal of Psychiatry Medicine 19: 181-192.

12.    WING RR and PHELAN S (2005) Long-term weight loss maintenance. American Journal of Clinical Nutrition 82: 222S-225S.

13.    Stote KS, Baer DJ, Spears K, Paul DR, Harris GK et al. (2007) A controlled trial of reduced meal frequency without caloric restriction in healthy, normal- weight, middle-aged adults. American Journal of Clinical Nutrition 85: 981-988.

14.    Johnstone A (2015a) Fasting for weight loss: an effective strategy or latest dieting trend?. International Journal of Obesity 39: 727-733.

15.    Osborne TB, Mendel LB, Ferry EL (1917) The Effect of Retardation of Growth Upon the Breeding Period and Duration of Life of Rats. Science 45: 294-295.

16.    Mattson MP, Duan W, Guo Z (2003) Meal size and frequency affect neuronal plasticity and vulnerability to disease: cellular and molecular mechanisms. Journal of Neurochemistry 84: 417-431.

17.    WEINDRUCH R and SOHAL RS (1997) Caloric Intake and Aging. New England Journal of Medicine 337: 986-994.

18.    Taormina G, MIrisola MG (2014) Calorie restriction in mammals and simple model organisms. Biomedical Research International 2014: 30869.

19.    Colman RJ, Anderson RM, Johnson SC, Kastman EK, Kosmatka KJ, et al. (2009) Caloric restriction delays disease onset and mortality in rhesus monkeys. Science 325: 201-204.

20.    Redman LM, Ravussin E (2011) Caloric restriction in humans: impact on physiological, psychological, and behavioural outcomes. Antioxidant Redox Signal 14: 275-287.

21.    Ye J, Keller JN (2010) Regulation of energy metabolism by inflammation: a feedback response in obesity and calorie restriction Aging 2: 361-368.

22.    Walford RL, Mock D, Verdery R, Maccallum T (2002) Calorie restriction in biosphere 2: alterations in physiologic, hematologic, hormonal, and biochemical parameters in humans restricted for a 2-year period. International Journal of Obesity 57: B211-B224.

23.    Lefevre M, Redman LM, Heilbronn LK, Smith JV, Martin CK, (2009) Caloric restriction alone and with exercise improves CVD risk in healthy non-obese individuals. Atherosclerosis 203: 206-213.

24.    Redman LM, Martin CK, Williamson DA, Ravussin E (2008) Effect of caloric restriction in non-obese humans on physiological, psychological and behavioural outcomes. Physiology and Behaviour 94: 643-648.

25.    Tsai AG, Wadden TA (2006) The evolution of very-low-calorie diets: an update and meta- analysis. Obesity (Silver Spring) 14: 1283-1293.

26.    Ryttig KR, Rossner S (1995) Weight maintenance after a very low-calorie diet (VLCD) weight reduction period and the effects of VLCD supplementation. A prospective, randomized, comparative, controlled long-term trial. Journal of Internal Medicine 238: 299-306.

27.    nakamura Y, Walker BR, Ikuta T (2016) Systematic review and meta-analysis reveals acutely elevated plasma cortisol following fasting but not less severe calorie restriction. Stress 19: 151-157.

28.    Vink RG, Roumans NJ, Arkenbosch LA, Mariman EC, Baak MAV (2016) The effect of rate of weight loss on long-term weight regain in adults with overweight and obesity. Obesity (Silver Spring) 24: 321-327.

29.    Rossner S, Flaten H (1997) VLCD versus LCD in long-term treatment of obesity. International Journal of Obesity Related Metabolic Disorders 21: 22-26.

30.    ROSSNER S, TORGERSON JS (2000) VLCD a safe and simple treatment of obesity. Lakartidningen 97: 3876-3879.

31.    SARIS WH (2001) Very‐Low‐Calorie Diets and Sustained Weight Loss. Obesity Research 9: 295S-301S.

32.    Jazet IM, DE Craen AJ, Schie EMV, Meinders AE (2007) Sustained beneficial metabolic effects 18 months after a 30-day very low-calorie diet in severely obese, insulin-treated patients with type 2 diabetes. Diabetes Research and Clinical Practice 77: 70-76.

33.    Speakman JR and Mitchell SE (2011) Caloric restriction. Molecular Aspects of Medicine 32: 159-221.

34.    Wadden TA, Stunkard AJ, Brownell KD, Day SC (1985) A comparison of two very-low-calorie diets: protein-sparing-modified fast versus protein-formula-liquid diet. American Journal of Clinical Nutrition 41: 533-539.

35.    Wadden TA, Stunkard AJ, Day SC, Gould RA, Rubin CJ (1987) Less food, less hunger: reports of appetite and symptoms in a controlled study of a protein-sparing modified fast. International Journal of Obesity 11: 239-249.

36.    weyer C, Walford RL, Harper IT, Milner M, Maccallum T, et al. (2000) Energy metabolism after 2 y of energy restriction: the biosphere 2 experiment. The American Journal of Medicine 72: 946-953.

37.    Walford RL, Weber L, Panov S (1995) Caloric restriction and aging as viewed from Biosphere 2. Receptor 5: 29-33.

38.    Palgi A, Read JL, Greenberg I, Hoefer MA, Bistrian BR, et al. (1985) Multidisciplinary treatment of obesity with a protein-sparing modified fast: results in 668 outpatients. American Journal of Public Health 75: 1190-1194.

39.    Racette SB, Weiss EP, Villareal DT, Arif H, Steger-May K, et al. (2006) One year of caloric restriction in humans: feasibility and effects on body composition and abdominal adipose tissue. American Journal of Clinical Nutrition 61: 943-950.

40.    Corral PD, Laney PC, Casazza K, Gower BA, Hunter GR (2009) Effect of dietary adherence with or without exercise on weight loss: a mechanistic approach to a global problem. Journal of Clinical Endocrinology and Metabolism 94: 1602-1607.

41.    Johnstone AM (2007) Fasting - the ultimate diet? Obesity Reviews 8: 211-222.

42.    Kostevski B (2012) The effects of intermittent fasting on human and animal health. Lund University 1: 130-165.

43.    Pilon B (2012) Eat Stop Eat, Strength Works International Publishing: 15-27

44.    Gotthardt JD, Verpeut JL, Yeomans BL, Yang JA, Yasrebi A, et al. (2016) Intermittent Fasting Promotes Fat Loss with Lean Mass Retention, Increased Hypothalamic Norepinephrine Content, and Increased Neuropeptide Y Gene Expression in Diet-Induced Obese Male Mice. Endocrinology 157: 679-691.

45.    Tinsley GM, LA Bounty PM (2015) Effects of intermittent fasting on body composition and clinical health markers in humans. Nutrition Reviews 73: 661-674.

46.    HORNE BD, MUHLESTEIN JB, ANDERSON JL (2015) Health effects of intermittent fasting: hormesis or harm? A systematic review. The Journal of the American Medical Association 102: 464-470.

47.    KAhleova H, Belinova L, Malinska H, Oliyarnyk O, Trnovska J, et al. (2014) Eating two larger meals a day (breakfast and lunch) is more effective than six smaller meals in a reduced-energy regimen for patients with type 2 diabetes: a randomised crossover study. Diabetologia 57: 1552-1560.

48.    Swindells YE, Holmes SA, Robinson MF (1968) The metabolic response of young women to changes in the frequency of meals. British Journal of Nutrition 22: 667-680.

49.    Antoine JM, Rohr R, Gagey MJ, Bleyer RE, Debry G (1984) Feeding frequency and nitrogen balance in weight-reducing obese women. Human Nutrition Clinical Nutrition 38: 31-38.

50.    Barnosky AR, Hoddy KK, Unterman TG, Varady KA (2014) Intermittent fasting vs daily calorie restriction for type 2 diabetes prevention: a review of human findings. Translational Research 164: 302-311.

51.    Ells LJ, Atkinson G, Mcgowan VJ, Hamilton S, Waller G, et al. (2015) Intermittent fasting interventions for the treatment of overweight and obesity in adults aged 18 years and over: a systematic review protocol. The JBI Database of Systematic Reviews and Implementation Reports 13: 60-68.

52.    Mattson MP and Wan R (2005a) Beneficial effects of intermittent fasting and caloric restriction on the cardiovascular and cerebrovascular systems. Journal of Nutritional Biochemistry 16: 129- 137.

53.    Klempel MC, Kroeger CM, Bhutani S, Trepanowski JF, Varady KA (2012a) Intermittent fasting combined with calorie restriction is effective for weight loss and cardio- protection in obese women. Nutrition Journal 11: 1-9.

54.    PIPER MDW and BARTKE A (2008) Diet and Aging. Cellular Metabolism 8: 99-104.

55.    Anson RM, Guo Z, Decabo R, Iyun T, Rios M, et al. (2003b) Intermittent fasting dissociates beneficial effects of dietary restriction on glucose metabolism and neuronal resistance to injury from calorie intake. Proceedings of the National Academy of Sciences 100: 6216-6220.

56.    Heilbronn LK, Smith SR, Martin CK, Anton SD, Ravussin E (2005b) Alternate-day fasting in no obese subjects: effects on body weight, body composition, and energy metabolism. American Journal of Clinical Nutrition 81: 69-73.

57.    Heilbronn LK, Civitarese AE, Bogacka I, Smith SR, Hulver M, et al. (2005a) Glucose tolerance and skeletal muscle gene expression in response to alternate day fasting. Obesity Research 13: 574-581.

58.    Varady KA (2011) Intermittent versus daily calorie restriction: which diet regimen is more effective for weight loss? Obesity Reviews 12: 593-601.

59.    Varady KA, Bhutani s, church EC, Klempel MC (2009) Short-term modified alternate-day fasting: a novel dietary strategy for weight loss and cardio protection in obese adults. American Journal of Clinical Nutrition 90: 1138-1143.

60.    Johnson JB, Summer W, Cutler RG, Martin B, Hyun DH D, et al. (2007) Alternate day calorie restriction improves clinical findings and reduces markers of oxidative stress and inflammation in overweight adults with moderate asthma. Free Radical Biology & Medicine 42: 665-674.

61.    Halberg N, Henriksen M, Soderhamn N, Stallknecht B, Ploug T, et al. (2005) Effect of intermittent fasting and refeeding on insulin action in healthy men. Journal of Applied Physiology 99: 2128-2136.

62.    Soeters MR, Lammers NM, Dubbelhuis PF, Ackermans M, Jonkers-Schuitema CF, et al. (2009) Intermittent fasting does not affect whole-body glucose, lipid, or protein metabolism. American Journal of Clinical Nutrition 90: 1244-1251.

63.    bellisle F, Mcdevitt R, Prentice AM (1997) Meal frequency and energy balance. British Journal of Nutrition 77: S57-70.

64.    Verboeket-Van DE Venne WP, Westerterp KR, Kester AD (1993) Effect of the pattern of food intake on human energy metabolism. British Journal of Nutrition 70: 103-115.

65.    Westerterp KR, Wilson SA, Rolland V (1999) Diet induced thermogenesis measured over 24h in a respiration chamber: effect of diet composition. International Journal of Obesity and Related Metabolic Disorders 23: 287-292.

66.    Sakamoto T, Takahashi N, Goto T, Kawada T (2014) Dietary factors evoke thermogenesis in adipose tissues. Obesity Research of Clinical Practice 8: e533-e539.

67.    Cardeal DAM, Faria SL, Faria OP, Facundes M, Ito MK (2016) Diet-Induced Thermogenesis in Post-Operative Roux-En-Y Gastric Bypass Patients with Weight Regain. Surgery for Obesity and Related Diseases 2: 302-345.

68.    Webber J, Macdonald IA (1994) The cardiovascular, metabolic and hormonal changes accompanying acute starvation in men and women. The American Journal of Medicine 71: 437- 447.

69.    gjedsted J, Gormsen LC, Nielsen S, Schmitz O, Djurhuus CB (2007) Effects of a 3-day fast on regional lipid and glucose metabolism in human skeletal muscle and adipose tissue. Acta Physiology 191: 205-16.

70.    Bryner RW, Ullrich IH, Sauers J, Donley D, Hornsby G, et al. (1999) Effects of resistance vs. aerobic training combined with an 800-calorie liquid diet on lean body mass and resting metabolic rate. The Journal of the American College of Nutrition 18: 115-121.

71.    Keim NL, Horn WF (2004) Restrained eating behaviour and the metabolic response to dietary energy restriction in women. Obesity Research 12: 141-149.

72.    wiesli P, Schwegler B, Schmid B, Spinas GA, Schmid C (2005) Mini-Mental State Examination is superior to plasma glucose concentrations in monitoring patients with suspected hypoglycaemic disorders during the 72-hour fast. European Journal of Endocrinology 152: 605-610.

73.    Alken J, Petriczko E, Marcus C (2008) Effect of fasting on young adults who have symptoms of hypoglycaemia in the absence of frequent meals. European Journal of Clinical Nutritio 62: 721-726.

74.    Awad S, Stephenson MC, Placidi E, Marciani l, teodosiu CD, et al. (2010) The effects of fasting and refeeding with a 'metabolic preconditioning' drink on substrate reserves and mononuclear cell mitochondrial function. Clinical Nutrition 29: 538-544.

75.    Rogers PJ, Smit HJ (2000) Food craving and food "addiction": a critical review of the evidence from a biopsychosocial perspective. Pharmacological Biochemistry and Behaviour 66: 3- 14.

76.    Kulovitz MG, Kravitz LR, Mermier C, Gibson AL, Conn CA, et al. (2014) Potential role of meal frequency as a strategy for weight loss and health in overweight or obese adults. Nutrition 30: 386-392.

77.    Perrigue MM, Drewnowski A, Wang CY, Neuhouser ML (2016) Higher Eating Frequency Does Not Decrease Appetite in Healthy Adults. Journal of Nutrition 146: 59-64.

78.    Grimm O (2007) Addicted to Food? Scientific American Mind 18: 36-39.

79.    Ozelli KL (2007) This is your brain on food. Scientific American 297: 84-85.

80.    Cummings DE and Overduin J (2007) Gastrointestinal regulation of food intake. Journal of Clinical Investigation 117: 13-23.

81.    Frecka JM and Mattes RD (2008) Possible entrainment of ghrelin to habitual meal patterns in humans. American Journal of Physiology and Gastrointestinal and Liver Physiology 294: G699- G707.

82.    Duncan GG (1962) Intermittent Fasts in the Correction and Control of Intractable Obesity. Transactions of the American Clinical and Climatological Association 74: 121-129.

83.    Speechly DP, Rogers GG, Buffenstein R (1999) Acute appetite reduction associated with an increased frequency of eating in obese males. International Journal of Obesity Related Metabolic Disorders 23: 1151-1159.

84.    Speechly DP, Buffenstein R (1999) Greater appetite control associated with an increased frequency of eating in lean males. Appetite 33: 285-297.

85.    Smeets AJ, Westerterp-Plantenga MS (2008) Acute effects on metabolism and appetite profile of one meal difference in the lower range of meal frequency. British Journal of Nutrition 99: 1316-1321.

86.    Munsters MJM, Saris WHM (2012) Effects of Meal Frequency on Metabolic Profiles and Substrate Partitioning in Lean Healthy Males. Plos One 7: e38632.

87.    Ohkawara K, Cornier MA, Kohrt WM, Melanson EL (2013) Effects of Increased Meal Frequency on Fat Oxidation and Perceived Hunger. Obesity (Silver Spring) 21: 336-343.

88.    Perrigue MM, Drewnowski A, Wang CY, Neuhouser ML (2016) Higher Eating Frequency Does Not Decrease Appetite in Healthy Adults. Journal of Nutrition 146: 59-64.

89.    Wan R, Camandola S, Mattson MP (2003) Intermittent fasting and dietary supplementation with 2-deoxy-D-glucose improve functional and metabolic cardiovascular risk factors in rats. The American Journal of Medicine 17: 1133-1134.

90.    Mager DE, Wan R, Brown M, Cheng A, Wareski P, et al. (2006) Caloric restriction and intermittent fasting alter spectral measures of heart rate and blood pressure variability in rats. Federation of American Societies for Experimental Biology 20: 631-637.

91.    Lee J, Herman JP, Mattson MP (2000) Dietary restriction selectively decreases glucocorticoid receptor expression in the hippocampus and cerebral cortex of rats. Experimental Neurology 166: 435-41.

92.    Roemmich JN, Sinning WE (1997) Weight loss and wrestling training: effects on growth- related hormones. Journal of Applied Physiology 82: 1760-1764.

93.    Soules MR, Merriggiola MC, Steiner RA, Clifton DK, Toivola B, et al. (1994) Short-term fasting in normal women: absence of effects on gonadotrophin secretion and the menstrual cycle. Clinical Endocrinology 40: 725-731.

94.    Gjedsted J, Gormsen L, Buhl M, Norrelund H, Schmitz O, Keiding S, et al. (2009) Forearm and leg amino acid metabolism in the basal state and during combined insulin and amino acid stimulation after a 3-day fast. Acta Physiology 197: 197-205.

95.    Jacoangel IF, Zoli A, Taranto A, Staar Mezzasalma F, Ficoneri C, et al. (2002) Osteoporosis and anorexia nervosa: relative role of endocrine alterations and malnutrition. Eating and Weight Disorders 7: 190-195.

96.    Bergendahl M, Vance ML, Iranmanesh A, Thorner MO, Veldhuis JD (1996) Fasting as a metabolic stress paradigm selectively amplifies cortisol secretory burst mass and delays the time of maximal nyctohemeral cortisol concentrations in healthy men. European Journal of Clinical Nutrition 81: 692-699.

97.    Larsen AE, Tunstall RJ, Carey KA, Nicholas G, Kambadur R, et al. (2006) Actions of short-term fasting on human skeletal muscle myogenic and atrogenic gene expression. Annals of Nutrition and Metabolism 50: 476-481.

98.    Veech RL, Chance B, Kashiwaya Y, Lardy HA, Cahill GF (2001) Ketone bodies, potential therapeutic uses. International Journal of Obesity 51: 241-247.

99.    Manninen AH (2004) Metabolic Effects of the Very-Low-Carbohydrate Diets: Misunderstood "Villains" of Human Metabolism. Journal of the International Society of Sports Nutrition 1: 7-11.

100. Rice B, Janssen I, Hudson R, Ross R (1999) Effects of aerobic or resistance exercise and/or diet on glucose tolerance and plasma insulin levels in obese men. Diabetes Care 22: 684- 691.

101. Janssen I, Fortier A, Hudson R, Ross R (2002a) Effects of an energy-restrictive diet with or without exercise on abdominal fat, intermuscular fat, and metabolic risk factors in obese women. Diabetes Care 25: 431-438.

102. Collier R (2013b) Intermittent fasting: the science of going without. Canadian Medical Association Journal 185: E363-E364.

103. Seimon RV, Shi YC, Slack K, Lee K, Fernando HA, et al. (2016) Intermittent Moderate Energy Restriction Improves Weight Loss Efficiency in Diet-Induced Obese Mice. Plos One 11: e0145157.

104. Harvie MN, Howell T (2016) Could Intermittent Energy Restriction and Intermittent Fasting Reduce Rates of Cancer in Obese, Overweight, and Normal-Weight Subjects? A Summary of Evidence. Advances in Nutrition 7: 690-705.

105. Johansson K, Neovius M, Hemmingsson E (2014) Effects of anti-obesity drugs, diet, and exercise on weight-loss maintenance after a very-low-calorie diet or low-calorie diet: a systematic review and meta-analysis of randomized controlled trials. The American Journal of Clinical Nutrition 99: 14-23.

106. Heymsfield SB, Greenberg AS, Fujioka K 1(999) Recombinant leptin for weight loss in obese and lean adults: A randomized, controlled, dose-escalation trial. The Journal of the American Medical Association 282: 1568-1575.

107. Jackson AS, Pollock ML (1978) Generalized equations for predicting body density of men. British Journal of Nutrition 40: 497-504.

108. Nevill AM, Metsios GS, Jackson AS, Wang J, Thornton J, et al. (2008) Can we use the Jackson and Pollock equations to predict body density/fat of obese individuals in the 21st century? International Journal of Body Composition Research 6: 114-121.

109. Tanner J, Whitehouse R (1955) The Harpenden skinfold caliper. American Journal of Physical Anthropology 13: 743-746.

110. slaughter M, Lohman T, Boileau R, Horswill C, Stillman R, et al. (2014) Skinfold equations for estimation of body fatness in children and youth. Human Biology 60: 4.

111. Michalsen A, Riegert M, Ludtke R, Backer M, Langhorst J, et al. (2005) Mediterranean diet or extended fasting's influence on changing the intestinal microflora, immunoglobulin A secretion and clinical outcome in patients with rheumatoid arthritis and fibromyalgia: an observational study. BMC Complementary and Alternative Medicine 5: 22.

112. Varady K, Bhutani S, Klempel M, Kroeger C (2011) Effect of alternate day fasting combined with exercise on body composition parameters in obese adults. Unpublished data.

113. Eshghinia S, Mohammadzadeh F (2013) The effects of modified alternate-day fasting diet on weight loss and CAD risk factors in overweight and obese women. Journal of Diabetes and Metabolic Disorders 12: 4-4.

114. Eshghinia S, Gapparov MG (2011) Effect of Short-Term Modified Alternate- Day Fasting on the Lipid Metabolism in Obese Women. Iranian Journal of Diabetes and Obesity 3: 1-5.

115. Johnstone AM, Faber P, Gibney ER, Elia M, Horgan G, et al. (2002) Effect of an acute fast on energy compensation and feeding behaviour in lean men and women. International Journal of Obesity Related Metabolic Disorders 26: 1623-1628.

116. Duncan G, Cristofori F, Yue J, Murthy M (1964) Control of obesity by intermittent fasts. Medical linics of North America 48: 1359.

117. Benedict FG, ROTH P (1918) Effects of a prolonged reduction in diet on 25 men: Influence on basal metabolism and nitrogen excretion. Proceedings of the National Academy of Sciences of the United States of America 4: 149-152.

118. Benedict FG (1918) Physiological effects of a prolonged reduction in diet on twenty-five men. Proceedings of the American Philosophical Society 57: 479-490.

119. STRANG JM and EVANS FA (1928) The energy exchange in obesity. Journal of Clinical Investigation 6: 277.

120. Walker WJ, Lawry EY, Love DE, Mann GV, Levine SA, et al. (1953) Effect of weight reduction and caloric balance on serum lipoprotein and cholesterol levels. The American journal of medicine 14: 654-664.

121. Atkinson RL, Kaiser DL (1985) Effects of calorie restriction and weight loss on glucose and insulin levels in obese humans. The Journal of the American College of Nutrition 4: 411-419.

122. Ballor DL, Katch VL, Becque MD, Marks CR (1988) Resistance weight training during caloric restriction enhances lean body weight maintenance. American Journal of Clinical Nutrition 47: 19-25.

123. Bales CW, Kraus WE (2013) Caloric restriction: implications for human cardiometabolic health. Journal of Cardiopulmonary Rehabilitation and Prevention 33: 201-208.

124. Pekkarinen T, Kaukua J, Mustajoki P (2015) Long-term weight maintenance after a 17-week weight loss intervention with or without a one-year maintenance program: a randomized controlled trial. Journal of Obesity 20: 651460.

125. Weiss EP, Jordan RC, Frese EM, Albert SG, Villareal DT (2016) Effects of weight loss on lean mass, strength, bone, and aerobic capacity. Medicine and Science in Sports and Exercise 1: 30-68.

126. Headland M, Clifton PM, Carter S, Keogh JB (2016) Weight-Loss Outcomes: A Systematic Review and Meta-Analysis of Intermittent Energy Restriction Trials Lasting a Minimum of 6 Months. Nutrients 8: E354.

127. Stamets K, Taylor DS, Kunselman A, Demers LM, Pelkman CL, et al. (2004) A randomized trial of the effects of two types of short-term hypocaloric diets on weight loss in women with polycystic ovary syndrome. Fertility and Sterility 81: 630-637.

128. Norrelund H, Borglum J, Jorgensen JO, Richelsen B, Moller N et al. (2000) Effects of growth hormone administration on protein dynamics and substrate metabolism during 4 weeks of dietary restriction in obese women. Clinical Endocrinology 52: 305-312.

129. Haugaard SB, Vaag A, Mu H, Madsbad S (2009) Skeletal muscle structural lipids improve during weight-maintenance after a very low calorie dietary intervention. Lipids and Health Disorders 8: 34.

130. Luscombe ND, Tsopelas C, Bellon M, Clifton PM, Kirkwood I (2006) Use of [14C]-sodium bicarbonate/urea to measure total energy expenditure in overweight men and women before and after low calorie diet induced weight loss. Asia Pacific Journal of Clinical Nutrition 15: 307-316.

131. Rickman AD, Williamson DA, Martin CK, Gilhooly CH, Stein RI, et al. (2011b) The CALERIE Study: Design and methods of an innovative 25% caloric restriction intervention. Contemporary Clinical Trials 32: 874-881.

132. Stewart TM, Bhapkar M, Das S, Galan K, Martin CK, et al. (2013) Comprehensive Assessment of Long-term Effects of Reducing Intake of Energy Phase 2 (CALERIE Phase 2) screening and recruitment: methods and results. Contemporary Clinical Trials 34: 10-20.

133. Fontana L, Villareal DT, DAS SK, SMITH SR, MEYDANI SN, et al. (2016) Effects of 2-year calorie restriction on circulating levels of IGF-1, IGF-binding proteins and cortisol in non-obese men and women: a randomized clinical trial. Aging Cell 15: 22-27.

134. Villareal DT, Fontana L, Das SK, Redman L, Smith SR, et al. (2016) Effect of Two-Year Caloric Restriction on Bone Metabolism and Bone Mineral Density in Non-Obese Younger Adults: A Randomized Clinical Trial. Journal of Bone and Mineral Research 31: 40-51.

135. Racette SB, Das SK, Bhapkar M, Hadley EC, Roberts SB, et al. (2012) Approaches for quantifying energy intake and calorie restriction during calorie restriction interventions in humans: the multicentre CALERIE study. American Journal of Clinical Nutrition 302: E441-E448.

136. Fontana L, Meyer TE, Klein S, Holloszy JO (2004) Long-term calorie restriction is highly effective in reducing the risk for atherosclerosis in humans. Proceedings of the National Academy of Sciences of the United States of America 101: 6659-6663.

137. Meyer TE, Kovacs SJ, Ehsani AA, Klein S, Holloszy JO, et al. (2006) Long-term caloric restriction ameliorates the decline in diastolic function in humans. Journal of the American College of Cardiology 47: 398-402.

138. Aranceta J and Pérez C (2006) Diario o registro dietético. Métodos de doble pesada. Nutrición y Salud Pública. Métodos, bases científicas y Aplicaciones 2: 158-67.

139. Wing RR, Blair E, Marcus M, Epstein LH, Harvey J (1994b) Year-long weight loss treatment for obese patients with type II diabetes: Does including an intermittent very-low- calorie diet improve outcome? The American Journal of Medicine 97: 354-362.

140. Seimon RV, Roekenes JA, Zibellini J, ZHU B, GIBSON AA, et al. (2015a) Do intermittent diets provide physiological benefits over continuous diets for weight loss? A systematic review of clinical trials. Molecular and Cellular Endocrinology, 418, Part 2, 153-172.

141. Davis CS, Clarke RE, Coulter SN, Rounsefell KN, Walker RE, et al. (2016) Intermittent energy restriction and weight loss: a systematic review. European Journal of Clinical Nutrition 70: 292-299.

142. Harvie MN, Pegington M, Mattson MP, Frystyk J, Dillon B, et al. (2011b) The effects of intermittent or continuous energy restriction on weight loss and metabolic disease risk markers: a randomized trial in young overweight women. International Journal of Obesity 35: 714-727.

143. Janssen I, Katzmarzyk PT, Ross R (2002b) Body mass index, waist circumference, and health risk: evidence in support of current National Institutes of Health guidelines. Archives of Internal Medicine 162: 2074-2079.

144. Martin B, Mattson MP, Maudsley S (2006) Caloric restriction and intermittent fasting: two potential diets for successful brain aging. Ageing Research Reviews 5: 332-353.

145. Gow ML, Garnett SP, Baur LA, Lister NB (2016) The Effectiveness of Different Diet Strategies to Reduce Type 2 Diabetes Risk in Youth. Nutrients 8: 194-230.

146. Dansinger ML, Gleason JA, Griffith JL, Selker HP, Schaefer EJ (2005) Comparison of the Atkins, Ornish, Weight Watchers, and Zone diets for weight loss and heart disease risk reduction: a randomized trial. Journal of the American Medical Association 293: 43- 53.

147. Summerbell CD, Cameron C, Glasziou PP (2008) Withdrawn: Advice on low-fat diets for obesity. Cochrane Database Systematic Reviews Cd003640.

148. Harvie M, Wright C, Pegington M, Mcmullan D, Mitchell E, et al. (2013) The effect of intermittent energy and carbohydrate restriction v. daily energy restriction on weight loss and metabolic disease risk markers in overweight women. British Journal of Nutrition, 110: 1534-1547.

149. Wegman MP, Guo MH, Bennion DM, Shankar MN, Chrzanowski SM, et al. (2015) Practicality of intermittent fasting in humans and its effect on oxidative stress and genes related to aging and metabolism. Rejuvenation Research 18: 162-172.

150. Collier R (2013a) Intermittent fasting: the next big weight loss fad. Canadian Medical Association Journal 185: E321-2.

151. Peiró PS, Sánchez MFA, Tejero SS (2012) La restricción calórica y el ayuno en la prevención y tratamiento del cáncer. Medicina Naturista 6: 22-32.

152. Harvie MN, Pegington M, Mattson MP, Frystyk J, Dillon B, et al. (2011a) The effects of intermittent or continuous energy restriction on weight loss and metabolic disease risk markers: a randomized trial in young overweight women. International Journal of Obesity 35: 460-498.

153. Franckle RL, Block JP, Roberto CA (2016) Calorie Underestimation When Buying High-Calorie Beverages in Fast-Food Contexts. American Journal of Public Health 106: 1254-1255.

154. Lansky D and Brownell Intermittent fasting: the next big weight loss fad KD (1982) Estimates of food quantity and calories: errors in self- report among obese patients. The American Journal of Clinical Nutrition 35: 727-732.

155. Heymsfield SB, Darby PC, Muhlheim LS, Gallagher D, Wolper C, et al. (1995) The calorie: myth, measurement, and reality. American Journal of Clinical Nutrition 62: 1034s-1041s.

156. Wansink B and Chandon P (2006) Meal size, not body size, explains errors in estimating the calorie content of meals. Annals of Internal Medicine 145: 326-332.

157. Lichtman SW, Pisarska K, Berman ER, Pestone M, Dowling H, et al. (1992) Discrepancy between self-reported and actual caloric intake and exercise in obese subjects. New England Journal of Medicine 327: 1893-1898.

158. Querol SE, Alvira JMF, Graffe MIM, Rebato NE, Sanchez AM, et al. (2016) Nutrient intake in Spanish adolescents SCOFF high-scorers: the AVENA study. Eating and Weight Disorders 2: 78-84.

159. Buschemeyer WC, Klink JC, Mavropoulos JC, Poulton SH, Wahnefried D, et al. (2010) Effect of intermittent fasting with or without caloric restriction on prostate cancer growth and survival in SCID mice. Clinical Nutrition 70: 138-173.

160. Lee K, Lee J, Bae WK, Choi JK, Kim HJ, et al. (2009) Efficacy of low-calorie, partial meal replacement diet plans on weight and abdominal fat in obese subjects with metabolic syndrome: a double-blind, randomised controlled trial of two diet plans - one high in protein and one nutritionally balanced. International Journal of Clinical Practice, 63: 195-201.

161. Tapsell L, Batterham M, Huang XF, Tan SY, Teuss G, et al. (2010) Short term effects of energy restriction and dietary fat sub-type on weight loss and disease risk factors. American Journal of Clinical Nutrition 20: 317-325.

162. Nicklas BJ, Wang X, You T, Lyles MF, Demons J, et al. (2009) Effect of exercise intensity on abdominal fat loss during calorie restriction in overweight and obese postmenopausal women: a randomized, controlled trial. American Journal of Clinical Nutrition 89: 1043-1052.

163. Das SK, Gilhooly CH, Golden JK, Pittas AG, Fuss PJ, Cheatham r A, et al. (2007) Long-term effects of 2 energy-restricted diets differing in glycemic load on dietary adherence, body composition, and metabolism in CALERIE: a 1-y randomized controlled trial. American Journal of Clinical Nutrition 85: 1023-1030.

164. Hill RJ and Davies PS (2001) The validity of self-reported energy intake as determined using the doubly labelled water technique. British Journal of Nutrition 85: 415-430.

165. Schoeller DA (1999) Recent advances from application of doubly labeled water to measurement of human energy expenditure. Journal of Nutrition 129: 1765-1768.

166. Livingstone MB and Black AE (2003) Markers of the validity of reported energy intake. Journal of Nutrition 133: 895s-920s.