Nutrition Society Summer Meeting 2016 held at University College Dublin on 11–14 July 2016

Conference on ‘New technology in nutrition research and practice’Postgraduate Symposium

Effects of intermittent fasting on glucose and lipid metabolism

Rona Antoni1*, Kelly L. Johnston2, Adam L. Collins1 and M. Denise Robertson11Department of Nutritional Sciences, University of Surrey, Guildford, Surrey, UK

2Department of Nutrition, LighterLife UK Ltd, Harlow, Essex, UK

Two intermittent fasting variants, intermittent energy restriction (IER) and time-restrictedfeeding (TRF), have received considerable interest as strategies for weight-managementand/or improving metabolic health. With these strategies, the pattern of energy restrictionand/or timing of food intake are altered so that individuals undergo frequently repeated per-iods of fasting. This review provides a commentary on the rodent and human literature, spe-cifically focusing on the effects of IER and TRF on glucose and lipid metabolism. For IER,there is a growing evidence demonstrating its benefits on glucose and lipid homeostasis in theshort-to-medium term; however, more long-term safety studies are required. Whilst themetabolic benefits of TRF appear quite profound in rodents, findings from the fewhuman studies have been mixed. There is some suggestion that the metabolic changes eli-cited by these approaches can occur in the absence of energy restriction, and in the contextof IER, may be distinct from those observed following similar weight-loss achieved viamodest continuous energy restriction. Mechanistically, the frequently repeated prolongedfasting intervals may favour preferential reduction of ectopic fat, beneficially modulateaspects of adipose tissue physiology/morphology, and may also impinge on circadianclock regulation. However, mechanistic evidence is largely limited to findings from rodentstudies, thus necessitating focused human studies, which also incorporate more dynamicassessments of glucose and lipid metabolism. Ultimately, much remains to be learned aboutintermittent fasting (in its various forms); however, the findings to date serve to highlightpromising avenues for future research.

Intermittent fasting: Glucose: Lipids: Weight-loss

We are currently in the midst of a global obesity epi-demic, with its prevalence more than doubling since1980(1). The development of obesity is closely associatedwith a number of metabolic complications includinginsulin resistance and dyslipidaemia, which can in turnincrease an individual’s risk of developing type 2 diabetesmellitus (T2DM) and CVD(2). Glucose and lipid homeo-stasis can be improved through modest weight-loss,which due to current accepted dietary advice, is mostcommonly achieved via a modest continuous (daily)energy restriction (CER)(3). Independently of weight-loss,the metabolic regulation of glucose and lipids can also beinfluenced by other dietary manipulations, includingalterations in meal timing(4) as well as abrupt changes

in energy status such as fasting, which when protracted,elicits profound shifts in fuel utilisation that persist dur-ing the subsequent refeeding period(5).

In recent years, several intermittent fasting variants,including intermittent energy restriction (IER) and time-restricted feeding (TRF) have received considerableinterest as alternative dietary strategies for weight-management and improving metabolic health. Withthese dietary strategies, the pattern of energy restrictionand/or timing of food intake are altered, so that indivi-duals undergo frequently repeated periods of fasting(Table 1). Their basic premise compared to CER isthat an individual may not need to energy restrict eitherevery day, or at all, to attain metabolic benefits. With

*Corresponding author: Dr R. Antoni, email r.antoni@surrey.ac.uk

Abbreviations: CER, continuous energy restriction; FAO, fatty-acid oxidation; FFA, free-fatty acid; HOMA-IR, homeostasis model assessment ofinsulin resistance; IER, intermittent energy restriction; T2DM, type 2 dibetes mellitus; TRF, time-restricted feeding.

Proceedings of the Nutrition Society (2017), 76, 361–368 doi:10.1017/S0029665116002986© The Authors 2017 First published online 16 January 2017

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these points in mind, the current review aims to provide acommentary on studies within the intermittent fasting lit-erature, with a specific focus on the effects of IER andTRF on glucose and lipid metabolism. Where possible,the relative contribution of weight-loss/energy restrictionand the intermittent fasting eating pattern per se toobserved metabolic changes will be discussed. In addition,the review highlights potential avenues for future research.

Intermittent energy restriction

Overview and effects on body weight

The majority of rodent studies(6–21) and a small numberof human studies(22–26) have used IER protocols, whichcompletely restrict energy intake (i.e. 100 % energyrestriction) every other day, with fasting intervals rangingbetween 20 and 36 h. However, the long-term sustain-ability of this alternate day total fasting approach inhuman subjects is questionable due to the persistent hun-ger reported(24). Subsequently, the IER protocols used bymost human studies(27–41), and by some rodent stud-ies(11,14,42), have allowed a small amount of ‘fast’ dayintake, so that energy is substantially (⩾70 %) but notcompletely restricted. This is often referred to as mod-ified fasting, such that, the term fasting in this IER con-text denotes periods of severe (total or partial) energyrestriction. These modified fasting forms of IER havepredominantly been utilised as weight-loss strategies,whilst a small number of studies have also used protocolsintended to encourage weight maintenance(37,39,41). Themost well-studied examples include alternate daymodified fasting and the 5:2 diet, which entails twomodified fast days a week (Table 1), although manyvariations exist. Intakes on non-restricted (‘feed’)days among these studies have ranged from adlibitum(27–29,31–34,36,38), hypoenergetic (approximately15–30 % of energy requirements)(34,41), isoener-getic(30,37,40) or hyperenergetic (approximately 125–175% of energy requirements)(35,39). Compliance is report-edly high(29,37), with both acute(5) and chronic(29,30,37,40)

studies demonstrating a lack of full compensatory hyper-phagia following modified fasting days. In overweight/obese(26–35,37,38,40,41) or combined healthy/overweight(36)

cohorts, the average reported weight-loss through IER(70–100 % energy restriction) has ranged betweenapproximately 4 and 10 % over dieting periods of 4–24weeks. There are also some data demonstrating the suc-cessful applications of IER as a weight-maintenance

strategy following weight-loss for periods of up to 1year(37,39).

Effects of intermittent energy restriction on glucose andlipid metabolism

Rodent studies. Although findings from rodent studiescannot be readily extrapolated to human subjects, they dopermit a greater level of control over dietary intakes andenvironmental confounders. Rodent studies of IER havemostly used alternate day total fasting protocols. Overstudy intervals of 4 weeks to 1 year, this form of IER canreduce levels of fasting glucose(6,8,9,21), insulin(8,9,16–18,21)

and insulin resistance (measured via homeostasis modelassessment of insulin resistance (HOMA-IR))(16,17), whichin turn reflects improved hepatic insulin sensitivity.Additionally, lipid profiles are also favourably altered inwild-type rodents(7,10,16), with similar observations madeby one alternate day modified (85 % energy restriction)fasting study(42). Findings from a number studies, whichhave performed more dynamic assessments of glycaemicindices, through use of gold-standard hyperinsulinaemic–euglycaemic glucose clamp techniques to assess insulinsensitivity or glucose/insulin tolerance testing, have beenmixed. Several studies have reported improvements inglycaemic control in rodents fed normal or low-fatchow(13,19), whilst some(17,20) (but not all(16)) studies haveshown that IER is able to protect against high-fatdiet-induced insulin resistance. In contrast, one studyconducted in obesity-prone Sprague–Dawley rats noted amarked impairment in glucose tolerance after 8 monthsof IER (normal chow)(15).

Human studies: alternate day total fasting. A small numberof studies have assessed the metabolic impacts of alternateday total fasting using a range of experimental techniques.Most recently, an 8-week pilot study completed bytwenty-six obese male and female participants reported anaverage weight-loss of 9 %, accompanied by beneficialreductions in LDL-cholesterol and fasting TAG(26). Theearlier 2(22,25)–3(23,24)-week studies conducted in smallgroups (n⩽ 16) of healthy and overweight participantswere not originally designed as weight-loss studies,although participants on the 3-week study struggled tosustain prescribed energy intakes on feed days, and soincurred a modest weight-loss(24). No post-treatmentchanges in fasting levels of insulin and glucose (whenmeasured after a feed day) were observed by these

Table 1. Overview of weekly fasting schedule for the most commonly studied intermittent fasting protocols. ‘Fast’ is used to denote periods ofsubstantial (total or partial, ⩾70%) energy restriction. Intermittent energy restriction (IER) protocols involving more modest energy restriction, or

IER–refeeding cycles not included in the review

Mon Tues Wed Thurs Fri Sat Sun

5:2 diet Fast Fast Feed Feed Feed Feed FeedAlternate day fasting Feed Fast Feed Fast Feed Fast FeedTime-restricted feeding ⩾12 h fast ⩾12 h fast ⩾12 h fast ⩾12 h fast ⩾12 h fast ⩾12 h fast ⩾12 h fast

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studies(22,23,25), whereas one study reported improvedlipid profiles(24). Two studies used hyperinsulinaemic–euglycaemic glucose clamp techniques to assess changesin glycaemic control, yielding conflicting results. Thehyperinsulinaemic–euglycaemic glucose clamp methodinvolves concomitant infusions of insulin (to achievehyperinsulinaemia) and glucose (to achieve a constantbasal level of plasma glucose). Under steady-stateeuglycaemic conditions, the glucose infusion rate equalsglucose uptake by all tissues and is therefore a measure oftissue sensitivity to exogenous insulin. Halberg et al.(22)

reported an increase (improvement) in insulin-mediatedwhole-body glucose uptake after 2 weeks in healthy men(BMI 25·7 (SD 0·4) kg/m2). In contrast, Soeters et al.(25),the only group among these earlier studies to have used a(cross-over) controlled study design, found no change ininsulin sensitivity in lean men following the same durationof time. In a 3-week study by Heilbronn et al.(23), whichwas conducted in a mixed sex healthy/overweight cohort,significant post-treatment reductions in postprandialinsulin responses to a test-meal challenge was notedamong male participants, whereas there was a contrastingdecline in glucose tolerance among female participants(with no change in the insulinaemic response). On closerexamination of the study design, post-treatment metabolicassessments were performed following a fast day (i.e. after36 h of fasting), whereas baseline assessments wereconducted after a shorter 12-h overnight fast. As such, theunstandardised fasting periods makes it difficult to ascribethese findings as a true chronic treatment effect, given thatprolonged fasting can also acutely alter postprandialsubstrate responses during the subsequent refeedingperiod(5). Ultimately, the uncontrolled study designsrepresent a significant limitation of most of these earlierstudies.

Human studies: modified fasting. To date, all IER studiesallowing fast day intake have solely measured fasting bloodmarkers of cardiometabolic risk. Some(27,30–33,35–37,41) butnot all of these studies(28,29,34,40) have included CER(standard treatment) or ad libitum (no intervention)control groups, whilst others have compared two ormore different IER protocols(35,38,39). In individualswith T2DM, IER has been shown to improveglycaemic control and lipid profiles(27). By contrast,many of the IER studies within heterogeneous non-T2DM populations, which have spanned from 4–24weeks, have failed to show any significant effect onfasting glucose levels(28,34,37–40) or glycated Hb(37);unsurprising findings given that glucose levels areusually kept under tight physiological control.However, improvements in other indices of insulinsensitivity have been observed, including reductions infasting insulin(30,37,39,40) and/or HOMA-IR(30,37,40). IERadditionally elicits favourable alterations in fasted lipidprofiles(28–33,35–38,41) including shifts in LDLsub-fraction distribution and/or size towards larger lessatherogenic particles(31–33,36,38), even in the absence ofoverall change in total LDL(38). Glycaemic andlipaemic outcomes do not appear to be influenced by

meal timing on modified fast days(38), or by dietarycomposition on feed days, although in this particularstudy vascular function worsened slightly when ahigh-fat (45 % total energy) diet was consumed(35). Inhealthy/overweight participants, Wegman et al.(39)

compared 3 weeks of IER with antioxidantsupplementation or placebo using a randomisedcross-over design. The protocol consisted of an energyintake of 25 and 175 % of energy requirements onalternating days, designed to maintain energy balance.The study observed a modest within-treatment decline infasting insulin following the IER plus placebointervention only, but did not conduct statisticalcomparisons between antioxidant and placebo legs.

Outstanding questions

Weight-management is an integral part of reducing car-diometabolic risk; even modest amounts of weight-losscan improve glycaemic control and lipid profiles, whichreflect improved glucose and lipid homeostasis. Theweight-loss efficacy of IER is not in doubt however,from a metabolic standpoint, a number of questionsremain outstanding:Can intermittent energy restriction influence

metabolism in the absence of overall energy restriction orweight-loss?. If IER could be shown to improvemetabolic control in the absence of overall energyrestriction or weight-loss, it would highlight additionalapplications of IER in healthy weight populations.There is some supportive evidence in the rodent literatureto suggest that IER is able to improve glycaemic controlmarkers, to a similar extent as CER (40 % energyrestriction), despite no overall energy deficit(8). Inhealthy/overweight human subjects, improvements ininsulin sensitivity have been observed after 2–3 weeksof alternate day total and modified fasting in theabsence of weight-change; however, these studies lackedappropriate ad libitum control group/interventions(22,39).There is clear paucity of data in this area and so morecontrolled studies in healthy weight populations arerequired to address this particular question.Does the mode of energy restriction (intermittent

v. continuous) influence metabolic responses duringweight-loss?. Ultimately it is the degree of dietaryadherence and sustainability, rather than the type ofdietary strategy, which will predict weight-loss outcomes.Understanding metabolic differences between dietingapproaches is nonetheless important as it may identifypotential applications of IER within certain patientsubgroups. Rodent studies (examining glycaemicindices) have demonstrated comparable short-termoutcomes between IER and CER(8,15,17), but also worselonger-term outcomes with IER(15), which is discussedfurther in the following section. Considering the rodentliterature in its entirety, a large proportion of studieshave reported a lack of full energy compensationon feed days, irrespective of background dietcomposition(9,10,13,16–19). Inclusion of a separatepair-fed group, i.e. a group fed an identical quantity offood but on a daily basis, is one way in which future

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studies could ascertain the relative contribution of theIER eating pattern and overall energy restriction toobserved metabolic changes.

Of the relatively few human studies to directly com-pare IER to CER, some studies have demonstrated com-parable metabolic outcomes between the two dietaryapproaches(26,27). The 5:2 diet studies, which were con-ducted in overweight/obese women, found reductions infasting insulin and HOMA-IR following IER that haveexceeded those of CER (25 % energy restriction/day)after 3 months(39) and 6 months(32), with both studiesreporting comparable average weight-loss between thetwo dieting groups. In a study by Varady et al.(32), thistime using an alternate day modified fasting regimen,IER led to a significant greater reduction in fastingTAG than CER after 12 weeks, with both dieting groupsattaining a comparable 5 % weight-loss.

From a metabolic perspective, without standardisationfor weight-loss between dieting groups, it becomes diffi-cult to interpret to what extent any underlying differencescan be explained simply by the extent of weight-loss.Whilst most dietary comparison studies have reportedsimilar average weight-loss between IER and CERgroups overall, the degree of weight-loss can vary consid-erably at an individual level, as exemplified by one suchstudy, which found a proportionally greater number ofIER participants tending to attain a ⩾5 % weight-loss(37).Notwithstanding, these data do highlight the potentialfor underlying differences between the two modes ofenergy restriction, which requires focused metabolicstudies, which conduct assessments following standar-dised weight-loss as opposed to diet duration.

By what mechanisms does intermittent energyrestriction alter metabolism, and how does this comparewith continuous (daily) energy restriction?Acute disturbances in fuel management. Over an acutetimescale, fasting (both total and modified) elicitsreciprocal shifts in fuel utilisation in a seemingly dose-responsive manner; free-fatty acid (FFA) mobilisation,fatty-acid oxidation (FAO) and ketogenesis progressivelyrise, favouring the conservation of glucose as its demanddrops(5). During the subsequent refeeding period after aprolonged (36 h) fast, postprandial fatty-acid partitioningremains shifted towards whole-body FAO and hepaticketogenesis, translating to a marked reduction inpostprandial lipaemia(5). From a chronic perspective,these acute disturbances in fuel management (experiencedrepeatedly during IER) may elicit very distinct alterationsin glucose and lipid metabolism when compared withCER, and may not necessitate an overall energyrestriction.For instance, the premise of IER is that the repeated

periods of FAO may promote or augment improvementsin insulin sensitivity via the preferential reduction in theectopic accumulation of lipid and associated cytosolicintermediaries, which are causally implicated in thedevelopment of insulin resistance(43); by extension, thiswould also be expected to favourably impact otheraspects of metabolism, such as the regulation of hepaticTAG metabolism(44). In support of this theory are

findings from the previously discussed human studiesdemonstrating superior reductions in HOMA-IR(30,37)

and fasting TAG(32) with IER v. CER, whilst anotherstudy found suggestions of increased mitochondrialfatty-acid transporter expression in skeletal muscle after3 weeks of IER, which reflects long-term adaptation tothe repeated elevations in FFA/FAO(23). In rodents, post-treatment reductions in hepatic steatosis have beenobserved(17,19), but the effects in human subjects remainsunknown.

Whilst diminished FAO capacity has been implicatedas a key instigator of insulin resistance, an alternativemitochondrial overload model by Koves et al.(45) pro-poses that increased FFA delivery is conducive to exces-sive and incomplete FAO and it is this that insteadprecipitates skeletal muscle insulin resistance. A majorcontributory factor is thought to be lipid-induced mito-chondrial stress, which is the primary site of endogenousreactive oxygen species production. In Koves’ model, theliver is considered relatively resilient to lipid-inducedmitochondrial stress due to its capacity to channel FFAtowards alternate metabolic pathways, with the keto-genic pathway being of particular relevance to fasting.Whilst Koves’ theory is framed in the context of obes-ity/high-fat feeding, it draws parallels with IER in thatmetabolic tissues are repeatedly exposed to elevationsin FFA/FAO with each fast–refeed cycle. Indeed thestudies (in both rodents and human subjects), whichreport impaired glucose tolerance, all of which haveused alternate day total fasting protocols, have alsofound increased markers of oxidative stress(15), oxidativeinsulin receptor modification(15), reduced GLUT con-tent(16), or indications of reduced mitochondrial functionmarkers(23) in skeletal muscle. Interestingly, insights intothe time-course of glycaemic changes were provided byone of the rodent studies, which found that despite aninitial improvement at 4 weeks, glucose tolerance thendeteriorated over time(15). These data highlight thepotential for harm, as well as the potential for tissue-specific responses to IER given that two of these studiesreported no change in fasting (hepatic) glycaemicindices(16,23).

Based on these data, we speculate that the alternateday total fasting paradigm may present too great or fre-quent metabolic challenge, which could potentially overtime lead to aberrant rather than beneficial changes inperipheral insulin sensitivity. In human subjects, theeffects of this form of IER on glycaemic control beyond8 weeks (where no change was observed) is unknown(26).Further research over longer timescales is now requiredto evaluate the potential harms and benefits of IER pro-tocols that in fast frequency and the severity of fast dayenergy restriction, as well as tissue-specific changes ininsulin sensitivity and ectopic lipid content. In addition,dynamic assessment of glucose and lipid metabolism,such as hyperinsulinaemic–euglycaemic glucose clamptechniques and postprandial challenges, may help todetect any underlying differences between IER andCER, which may not be immediately apparent whensolely looking at singular blood measurements taken inthe fasted state.

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Change in adipose tissue morphology, physiology anddistribution

Adipose tissue is integral to the maintenance of glucoseand lipid homeostasis; not only does it act as importantsink and storage organ for FFA, but also as a sourceof an array of adipokines capable of influencingwhole-body metabolism. An important driver of obes-ity-associated metabolic disorders appears to be thepathological expansion of adipose tissue among predis-posed individuals, the manifestations of which include:impaired adipogenesis and excessive adipocyte hyper-trophy in subcutaneous depots; macrophage infiltration,resulting in low grade inflammation and altered adipo-kine secretion; impaired insulin action and dysregulatedlipid storage; visceral adipose tissue accumulation. Theresult is the creation of an unfavourable metabolic andhormonal milieu, which drives the development of sys-temic insulin resistance and associated metabolicdisorders(46).

Several studies have shown that rodents maintained onIER exhibit reduced visceral adipose tissue(14,42), as wellas favourable reductions in fat cell size and augmentedadipocyte differentiation within subcutaneous and vis-ceral fat pads(11). Levels of adiponectin, which has anti-atherogenic and insulin-sensitising properties, are usuallyreduced in states of obesity but increased throughIER(14,21). On the basis of one study, these beneficialchanges in adipose tissue physiology and morphologyappear to be comparable with CER(14), but what wasunique to IER is that that it did not necessitate an overallenergy deficit, which is perhaps attributable to the alter-nating periods of feeding and fasting. In human subjects,one uncontrolled alternate day total fasting foundimproved insulin-mediated lipolysis inhibition, reflectingimproved insulin action, after 2 weeks(22). In addition toreductions in total adiposity, IER weight-loss studieshave reported reductions in visceral adipose tissue(38),truncal adiposity(26) as well as beneficial changes in circu-lating adipokine profiles. For example, pro-atherogenicadipokines, including leptin(26,30,36,37) are reduced, whilstadiponectin levels are increased(30,36). Among studiescomparing changes in adiposity measures (total, truncaland waist circumference)(26,27,30,37) and/or circulatingadipokines(26,30,37) between IER and CER, no consistentdifferences have been observed; however, additionalstudies employing robust assessments of regional adipos-ity are needed.

Time-restricted feeding

Overview

Over the past few years, there has been an emergence ofinterest in the concept of chrononutrition, i.e. the inter-action between meal timing and our circadian system,which comprise self-sustained approximately 24 h oscil-lations in physiology, metabolism and behaviour(47).Examples include the daily rhythms in glucose homeosta-sis and insulin sensitivity, which declines over the courseof the day(48). These rhythms are driven by a series of

molecular ‘clocks’(47). The ‘master’ clock is located in asmall brain region within the anterior hypothalamus,the suprachiasmatic nuclei. Light is the dominant entrai-ner (Zeitgeber) of the suprachiasmatic nuclei, which con-trols many essential physiological processes, includingthe sleep–wake cycle and endocrine rhythms(47).Peripheral clocks are located in many tissues integral toglucose and lipid metabolism such as the liver, pancreas,skeletal muscle and adipose tissue. Feeding time acts asthe major Zeitgeber of peripheral clocks, with the supra-chiasmatic nuclei acting as the ‘central conductor’, ensur-ing correct synchronisation between peripheral clocks(48).Possession of such rhythms permits effectiveco-ordination of endogenous processes to changes inthe environment such as the daily light–dark cycle, andconsequent cyclical food availability. TRF is oneexample of a timed dietary approach, which also fallsunder the intermittent fasting umbrella (Table 1). TRFinvolves limiting food intake to within a shortened win-dow of time, which thereby extends the length of thedaily fasting interval. In contrast to IER, TRF is usuallyperformed on a daily basis and does not necessarily entaila prescribed energy restriction; however, the twoapproaches are not mutually exclusive, indeed, alternateday total fasting can be seen as a prolonged form ofTRF. An overview of findings from the rodent andhuman literature is discussed next.

Effects of time-restricted feeding on glucose and lipidmetabolism

Rodents. Being nocturnal animals, mice consume themajority of their energy during the dark/active phase.TRF (⩽12 h) where food intake is consolidated to thedark phase, has been shown to both protect againstand reverse the harmful metabolic consequences ofdiverse nutritional challenges, including high-fat andhigh-sugar obesogenic diets(49,50). TRF mice displayreduced adiposity and liver steatosis, as well asimproved glucose tolerance and reduced cholesterollevels when compared with mice fed ad libitum with thesame high-fat diet; these changes may reflect improvedhomeostasis in multiple tissues(49,50). Importantly, theseimprovements occur in the absence of changes inenergy intake.Human studies. In human individuals, TRF has been

achieved through avoidance of night-time intake(51), bylimiting food intake to one evening meal daily (referredto herein as evening TRF)(52,53), or by allowingparticipants to self-select their own shortened (10–11 h)eating window(54). Islamic religious fasting restrictsfood access to nocturnal hours, between sunrise andsunset. Recent meta-analyses of these Ramadanstudies, which by their nature have been observationalby design, demonstrate improvements in somebiochemical risk markers despite the nocturnal eatingpattern(55). With regard to interventional TRF studies,only two publications derived from the same cross-overcontrolled study conducted within a healthy-weightmiddle-aged study cohort, have reported metabolicoutcomes. Stote et al.(53) found both pro-atherogenic

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(increasing LDL-cholesterol) and anti-atherogenic(increasing HDL-cholesterol and decreasing TAG)changes following 8 weeks of evening TRF. Findingsmay have been confounded by circadian variations inthese parameters, as baseline and post-interventionblood measurements were taken in the morning andevening, respectively. In the second publication, thegroup also reported significant elevations in fasting andpostprandial glycaemia(52). In this instance, oral glucosetolerance testings were conducted in the morning,following a period of time in which participants mayhave become metabolically adapted to this eveningeating pattern. In addition, as there was nostandardisation of dietary intakes on the day precedingtheir two metabolic assessments, participants wouldhave consumed a greater quantity of food the nightbefore their post-intervention visit, which might explainthe rise in fasting glucose relative to baseline.

Outstanding questions

Whilst pervasive rodent data exist highlighting the sub-stantive health benefits of TRF, human data are sparseand their interpretation hindered by methodologicalissues. As such, a number of fundamental questionsremain outstanding:

By what mechanisms might time-restricted feedingreduce disease risk, and to what extent can anymetabolic benefits be attributable to fasting?.Misalignment between our circadian biology andbehavioural patterns, for example through consumptionof food outside of the appropriate phase of theendogenous circadian cycle, is thought to be a majorcontributor to obesity and metabolic disease risk(48).Defined daily feeding and fasting rhythms supportrobust oscillation in components of the circadian clockand their output genes, but become dampened inrodent models of diet induced obesity as they start toconsume a greater proportion of their food intake inthe light (inactive) phase(49,50). Rhythmicity can berestored in these rodent models through TRF, which ismade possible by the extensive cross-talk andinteraction between molecular clock components andthe feed/fasting responsive signals of cellular energystatus. For example, fasting induces the activities of theAMP-activated protein kinase and cAMP-responseelement-binding protein, which promote ATP productionduring low-energy availability. In contrast, feedingstimulates the mechanistic target of rapamycin pathway,which promotes anabolic processes during increasedenergy availability. These feed/fast responsive elementscan influence the expression of peripheral clockcomponents and vice versa(56). In support of this, TRFhas been shown to improve cAMP-response element-binding protein, mechanistic target of rapamycin, andAMP-activated protein kinase rhythms, and hencepromote robust oscillations of circadian clock genecomponents(49). The metabolic benefits of TRF mayadditionally be attributed to the accompanyingelongation of daily fasting intervals, which as previouslydiscussed, may favourably impact on regional and

ectopic adiposity. In the comprehensive rodent studyconducted by Chaix et al.(50), the magnitude of themetabolic benefits of TRF were proportional to thelength of the fasting interval. Whilst highly suggestive,there are no comparable human data. In addition, whilstone purported benefit of TRF is that it constrains energyintake to the most appropriate phase of the endogenouscircadian cycle, in human subjects, the impact of earlierTRF meal timings (when metabolic control is moreoptimal(48)), has yet to be assessed.Can time-restricted feeding alter metabolism in the

absence of energy restriction in human subjects?. Onemajor difference between human and rodent studies isthat human subjects tend to behaviourally reduce foodintake during TRF when permitted ad libitum/self-selected intake(51,54). Similarly, many Ramadanstudies report a mild energy deficit, although this is not auniversal finding(55,57). Where energy restriction is absent,or minimal, current evidence suggests that TRF caninfluence metabolism (both positively and negatively),necessitating further study into the optimal fastingwindow(52,53). Although this often unintentional energyrestriction makes metabolic interpretations difficult, thesignificance cannot be disregarded as it highlights thepotential utility of TRF as a ‘small changes’ strategy forweight management. However, from a metabolicstandpoint, further studies enforcing tighter control andmonitoring over both energy intake and expenditurewould be needed to fully explore this.

Conclusion

Put together, there is a growing evidence base demon-strating the benefits of IER over the short-to-mediumterm on glucose and lipid homeostasis, meriting furtherresearch in population groups with (or are at high riskof) T2DM and CVD, including healthy-weight indivi-duals. Whilst the metabolic benefits of TRF appearquite profound in rodents, studies in human subjectsare sparse and subject to some methodological issues,which hinder their interpretation. There is some sugges-tion that the metabolic changes elicited by the two inter-mittent fasting approaches can occur in the absence ofenergy restriction, and in the context of IER, may be dis-tinct to those observed following similar weight-lossachieved via modest CER. Specifically, some studieshave found greater improvements in indices of hepaticinsulin sensitivity and TAG metabolism with IER. Themetabolic benefits of intermittent fasting are likely tostem in part from the frequently repeated prolonged fast-ing intervals, which may favour the preferential reduc-tion of ectopic fat, beneficially modulate aspects ofadipose tissue physiology/morphology and distribution(in addition to any changes in overall mass), and mayalso impinge on circadian clock regulation. Howevermechanistic evidence is largely limited to findings fromrodent studies, thus necessitating focused, controlled,human studies, which also incorporate more dynamicassessments of glucose and lipid metabolism in additionto steady-state measurements. Long-term studies are

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also needed, which may yet uncover the potential foradverse health consequences, as evidenced by a smallnumber of studies. Ultimately, much remains to belearned about intermittent fasting (in its various forms);however, the positive findings to date serve to highlightpromising avenues for future research.

Financial Support

R. A. was funded jointly by the University of SurreyPrize studentship and by Lighterlife

Conflicts of Interest

K. L. J. is the Head of Nutrition and Research atLighterlife.

Authorship

R. A. wrote the manuscript. K. L. J., A. L. C. andM. D. R. assisted with manuscript editing.

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