Abstract Our current 24-h society requires an increasing number of employees to work nightshifts with millions of people worldwide working during the evening or night. Clear associations have been found between shiftwork and the risk to develop metabolic health problems, such as obesity. An increasing number of studies suggest that the underlying mechanism includes disruption of the rhythmically organized body physiology. Normally, daily 24-h rhythms in physiological processes are controlled by the central clock in the brain in close collaboration with peripheral clocks present throughout the body. Working schedules of shiftworkers greatly interfere with these normal daily rhythms by exposing the individual to contrasting inputs, i.e., at the one hand (dim)light exposure at night, nightly activity and eating and at the other hand daytime sleep and reduced light exposure. Several different animal models are being used to mimic shiftwork and study the mechanism responsible for the observed correlation between shiftwork and metabolic diseases. In this review we aim to provide an overview of the available animal studies with a focus on the four most relevant models that are being used to mimic human shiftwork: altered timing of (1) food intake, (2) activity, (3) sleep, or (4) light exposure. For all studies we scored whether and how relevant metabolic parameters, such as bodyweight, adiposity and plasma glucose were affected by the manipulation. In the discussion, we focus on differences between shiftwork models and animal species (i.e., rat and mouse). In addition, we comment on the complexity of shiftwork as an exposure and the subsequent difficulties when using animal models to investigate this condition. In view of the added value of animal models over human cohorts to study the effects and mechanisms of shiftwork, we conclude with recommendations to improve future research protocols to study the causality between shiftwork and metabolic health problems using animal models. Introduction Our current 24-h society requires an increasing number of employees to work nightshifts and as a result millions of people worldwide work during the evening or night for a certain period during their life. In the Netherlands, 16% of the working population works regularly or occasionally during the night, whereas 51% of the population sometimes or regularly works during the evening (Centraal Bureau voor de Statistiek, 2013) . Epidemiological studies show correlations between shiftwork and an increased risk of cancer, cardiovascular disease, sleep disturbances, impaired psychosocial health and gastrointestinal problems (Matheson et al., 2014) . Moreover, the last two decades, population-based studies have shown that there is also an association between shiftwork and development of metabolic problems, including metabolic syndrome (Van Amelsvoort et al., 1999; Karlsson et al., 2001 Karlsson et al., , 2003 Biggi et al., 2008; Suwazono et al., 2008; Lin et al., 2009; Pietroiusti et al., 2010; Kubo et al., 2011; Li et al., 2011; Tucker et al., 2012; Ye et al., 2013; Kawabe et al., 2014; Kawada and Otsuka, 2014) , altered glucose metabolism (De Bacquer et al., 2009; Suwazono et al., 2009; Oyama et al., 2012) , altered lipid metabolism (Biggi et al., 2008; De Bacquer et al., 2009; Dochi et al., 2009) and high blood pressure (Morikawa et al., 1999; Sakata et al., 2003; De Bacquer et al., 2009; Lin et al., 2009) . Population-based studies are limited in their use for understanding causality and underlying mechanisms to explain the relationship between shiftwork and disease. Using experimental studies in humans is problematic due to the fact that many metabolic outcomes, such as body weight and composition, are long-term effects. Certainly, acute effects of shiftwork conditions on metabolic parameters can be studied in humans, which is currently done (McHill et al., 2014) . Therefore, animal studies have been used to gain more insight in these questions. In the current review we provide an overview of the different animal models that are available to investigate the mechanism underlying the negative health consequences of shiftwork. Introduction Daily 24-h rhythms are present throughout the body's physiology and can be observed in, for example, sleep, food consumption, body temperature and numerous hormone levels (Dibner et al., 2010) . These rhythms are regulated by the central circadian clock in the suprachiasmatic nucleus (SCN) of the hypothalamus FIGURE 1 | Shiftwork can be disentangled into different aspects (blue blocks), for some of these aspects animal models have been developed (green blocks). Each of these aspects might contribute to health risks associated with shiftwork. However, all aspects strongly interact, making it difficult to separate the effects of each single aspect. In most animal studies only one of the aspects is manipulated, however, it is important to keep in mind that by manipulation of one aspect, other aspects might be affected as well due to this interaction. and circadian oscillators in peripheral tissues and organs (the so-called peripheral clocks). The endogenous rhythmicity of the SCN neurons ultimately results from the interaction between a set of rhythmically expressed genes, so-called clock genes, which are expressed in almost every cell of the body. In the SCN, the nearly 24-h (i.e., circadian) rhythms produced by these clockgenes are synchronized to the exact 24-h rhythms in the outer world by their sensitivity to (sun)light (Dibner et al., 2010) . The synchronizing stimuli for peripheral clocks in non-SCN tissues are less clear, in addition to nervous and humoral signals from the SCN, behavioral signals such as body temperature, energy metabolism and (feeding) activity also likely play a role (Hastings et al., 1998; Dibner et al., 2010) . Introduction In general, the working schedules of shiftworkers profoundly interfere with these normal daily rhythms (Puttonen et al., 2010; Fritschi et al., 2011) . Shiftwork leads to a disruption of the circadian rhythms produced by the central and peripheral clocks by confronting them with opposing signals, i.e., light at night and food consumption and activity during the sleep period. Therefore, shiftwork is a challenge that contains many aspects, which might be related to the negative health effects (Figure 1 ): (1) social pattern: shiftwork affects social life due to working hours that conflict with working hours of social contacts; (2) activity: shiftwork affects the timing of people's activity and, as a consequence, possibly affects the amount of activity; (3) sleep: shiftwork affects timing of sleep and possibly duration and quality of sleep; (4) nutrition: shiftwork affects timing of food intake and possibly meal frequency and composition; (5) light exposure: shiftwork affects the timing of light exposure, with possibly different intensity and duration of exposure; (6) sun exposure: shiftwork might affect the duration of sun exposure and as a consequence vitamin D levels. Shiftwork comprises alterations at different levels of the circadian system that each have their own effect, but are interacting as well (Figure 1 ; Puttonen et al., 2010; Fritschi et al., 2011) . For example, the altered timing of activity in shiftworkers may result in sleep disturbances (duration, quality, and timing), altered nutrition (composition, caloric intake and timing), changed lighting exposure conditions (duration, intensity, and timing), reduced sunlight exposure (possible effect on vitamin D levels) and disturbances in social life. Each of these aspects might, to a greater or lesser extent, contribute to negative health effects. For several aspects, animal models have been developed to examine the metabolic health effects upon manipulation of these aspects of shiftwork individually or in combination (Figure 1) . To our knowledge, no animal models have so far been developed to study effects of "social life" and "sun exposure." Introduction The aim of the present review is to provide an overview of the available animal studies investigating the mechanism underlying the negative health consequences of shiftwork and their outcome. In addition, we discuss human relevancy of the available animal models for shiftwork to gain insight into animal to human translatability and aid future investigations in choosing optimal animal models. Next to animal models mimicking circadian disruptive shiftwork aspects, consequences of circadian disruption have also been studied in animals using genetic manipulation or SCN lesions. These animal models are not within the scope of the present review, since we do not consider them to represent human shiftwork. Introduction To increase the animal to human translatability the focus of the present review is on animal models investigating the relationship between shiftwork and metabolic risk factors (Haffner, 1998; Carnethon et al., 2004; Esquirol et al., 2011) since these factors are easily translatable from humans to animals and vice versa. In addition, these factors can be measured almost non-invasively in humans and often appear before the full blown disease, allowing for shorter follow-up time and more time for preventive measures or intervention. Introduction Studies were included in the review when they investigated metabolic parameters such as bodyweight, food intake, activity, glucose metabolism (including plasma glucose, insulin and glucagon levels, glucose tolerance, and glycogen levels), leptin levels, and lipid metabolism (including plasma cholesterol and triglyceride levels). Methods Search Strategy A literature search was performed to obtain an overview of the current scientific literature on studies using animal models for shiftwork to investigate the relationship between shiftwork and metabolic function. The search strategy was designed by an information specialist (RIVM) using the MESH-database of Pubmed, to include all MeSH terms and its synonyms and several electronic databases were used (Medline, Embase, BIO-SIS Previews en SciSearch). In brief, the search strategy combined keywords related to shiftwork with keywords related to metabolic risk factors. Examples of key words for shiftwork: shift work * , shiftwork * , night work * , night shift * , rotating shift * , jet lag, working rhythm * , "irregular working hours, " time restricted, "constant light, " "continuous light, " "light at night, " biological clock * , body clock * , chronobiology * , circadian clock * . Examples of key words for metabolic risk factors: weight, body weight, weight change, metabolic syndrome, obesity, adiposity, glucose, glucose tolerance, lipid metabolism, energy metabolism, insulin, insulin sensitivity, hypertension, leptin. Only papers published after 1993 were included. For the complete search strategy see Supplementary Data. Search Strategy The search resulted in 1550 publications, but only 44 were included as these met the following criteria: Search Strategy (1) Using animal models for shiftwork (2) Investigate effects on at least one of the following metabolic risk factors for disease (Haffner, 1998; Carnethon et al., 2004) : Search Strategy bodyweight and related measures (BMI, fat percentage) or glucose homeostasis: including plasma glucose levels, glucose tolerance, plasma insulin levels, insulin sensitivity or lipid homeostasis: including plasma levels of triglycerides, cholesterol, free fatty acids, HDL or LDL. The search included papers in English, Dutch, French, and German. However, only papers in English fitted the above mentioned criteria. In addition to this search strategy, the present knowledge of the authors and references from included papers ("snowball method") were used to include additional papers that fitted the above mentioned criteria (including papers published before 1993) or investigated parameters related to metabolic dysfunction. Categorization of Studies Included studies were divided in four different categories as presented in Figure 1: (1) Models using "timing of food intake, " (2) models using "timing of activity, " (3) models using "timing of sleep, " and (4) models using "timing of light." For each study the outcome parameters were determined, which included the abovementioned metabolic risk factors for disease as well as circadian parameters (e.g., activity, cortisol) and gene expression. For these parameters, results are described in the text and summarized in the table for overview purposes. In the Tables 1-5 , the left column holds the metabolically relevant parameters which were scored for and were most frequently measured in the studies. The rat and mouse columns represent the number of studies in which an effect of the manipulation (compared to the control condition) was found in this species against the number of studies in which it was measured. In the total column, results are divided in direction of effects and presented as the number of studies observing that direction is summarized. This was not done for gene expression. The most right column shows the studies in which the parameter was measured in this category of models. In the tables, studies are counted twice when multiple experiments are performed in one article, for instance when two types of mice were used. Occasionally, the effect of the manipulation was measured on the total level as well as the rhythm of a parameter within the same study. In this case, both effects are included in the total column. Categorization of Studies With these tables we aim to provide an overview of the metabolic parameters that are influenced per category and type of animals used. Results of effect on gene expression are described in the text and summarized in the table as "gene expression." The present review was aimed at providing a narrative overview of Arble et al., 2009; Bray et al., 2010 Bray et al., , 2013 Salgado-Delgado et al., 2010a , 2013 Jang et al., 2012; Sherman et al., 2012; Yoon et al., 2012; Reznick et al., 2013; Oyama et al., 2014; Shamsi et al., 2014 Food intake total 1/3 4/8 +: 0/11 (0%) −: 5/11 (45, 5%) o: 6/11 (54, 5%) Arble et al., 2009; Bray et al., 2010 Bray et al., , 2013 Salgado-Delgado et al., 2010a , 2013 Jang et al., 2012; Sherman et al., 2012; Yoon et al., 2012; Reznick et al., 2013; Oyama et al., 2014; Shamsi et al., 2014 Activity total 1/2 2/5 +: 2/7 (28, 6%) −: 1/7 (14, 3%) o: 4/7 (57, 1%) Arble et al., 2009; Bray et al., 2010 Bray et al., , 2013 Salgado-Delgado et al., 2010a; Sherman et al., 2012; Yoon et al., 2012; Reznick et al., 2013; Shamsi et al., 2014 EE total 1/1 2/2 +: 0/3 (0%) −: 3/3 (100%) o: 0/3 (0%) Arble et al., 2009; Bray et al., 2010 Bray et al., , 2013 Reznick et al., 2013 RER 1/1 2/2 +: 1/3 (33, 3%) −: 1/3 (33, 3%) ∼: 1/3 (33, 3%) o: 0/3 (0%) Bray et al., 2010 Bray et al., , 2013 Reznick et al., 2013 Adiposity 2/3 3/4 +: 3/7 (42, 8%) −: 2/7 (28, 6%) o: 2/7 (28, 6%) Arble et al., 2009; Bray et al., 2010; Salgado-Delgado et al., 2010a , 2013 Sherman et al., 2012; Reznick et al., 2013; Shamsi et al., 2014 Glucose metabolism 2/3 4/6 +: 1/9 (11, 1%) −: 2/9 (22, 2%) ∼: 3/9 (33, 3%) o: 3/9 (33, 3%) Bray et al., 2010 Bray et al., , 2013 Salgado-Delgado et al., 2010a , 2013 Jang et al., 2012; Sherman et al., 2012; Yoon et al., 2012; Reznick et al., 2013; Shamsi et al., 2014 Lipid metabolism 2/2 3/5 +: 1/7 (14, 3%) −: 2/7 (28, 6%) ∼: 4/7 (57, 1%) o: 2/7 (28, 6%) Bray et al., 2010 Bray et al., , 2013 Salgado-Delgado et al., 2010a; Sherman et al., 2012; Yoon et al., 2012; Reznick et al., 2013; Shamsi et al., 2014 Corticosterone 2/2 2/3 +: 1/5 (20%) −: 0/5 (0%) ∼: 3/5 (60%) o: 1/5 (20%) Salgado-Delgado et al., 2010a; Sherman et al., 2012; Bray et al., 2013; Reznick et al., 2013; Shamsi et al., 2014 Melatonin Leptin 1/1 0/1 +: 1/2 (50%) −: 0/2 (0%) ∼: 1/2 (50%) o: 1/2 (50%) Bray et al., 2010; Sherman et al., 2012; Reznick et al., 2013 Ghrelin 1/1 Categorization of Studies BP/Heart rate 2/2 +: 0/2 (0%) −: 2/2 (100%) ∼: 0/2 (0%) o: 0/2 (0%) Schroder et al., 2014 Gene expression 2/2 10/10 12/12 (100%) Damiola et al., 2000; Jang et al., 2012; Sherman et al., 2012; Yoon et al., 2012; Bray et al., 2013; Reznick et al., 2013; Salgado-Delgado et al., 2013; Oyama et al., 2014; Shamsi et al., 2014 For detailed description of the columns, see Methods section. Murphy, 2003; Tsai and Tsai, 2007; Salgado-Delgado et al., 2008 , 2010a , 2013 Leenaars et al., 2012 Food intake total 2/6 +: 1/6 (16, 7%) −: 1/6 (16, 7%) o: 4/6 (66, 7%) Murphy, 2003; Tsai and Tsai, 2007; Salgado-Delgado et al., 2008 , 2010a ,b, 2013 Activity total 4/6 +: 0/6 (0%) −: 4/6 (66, 7%) o: 2/6 (33, 3%) Salgado-Delgado et al., 2008 , 2010a ,b, 2013 Leenaars et al., 2012; Hsieh et al., 2014 EE total RER Adiposity 2/2 +: 2/2 (100%) −: 0/2 (0%) o: 0/2 (0%) Salgado-Delgado et al., 2010a , 2013 Glucose metabolism 3/3 +: 1/3 (33, 3%) −: 2/3 (66, 7%) ∼: 0/3 (0%) o: 0/3 (0%) Salgado-Delgado et al., 2008 , 2010a , 2013 Lipid metabolism 2/2 +: 0/2 (0%) available studies and their findings, due to heterogeneity studies were not assessed for quality. Results Animal studies that model human shiftwork can be divided into four main categories. The first three categories are based on desynchronization of peripheral clocks from the central clock by the unnatural timing of food intake, sleep or activity. The remaining category consists of studies that manipulate the timing of light exposure, including alterations of duration (i.e., continuous light) and timing of light exposure. Some studies used a shiftwork model that combines multiple categories and those will be mentioned repeatedly in the different categories if appropriate. Below we discuss the main findings of studies using a shiftwork model within the categories where the models fit best. Results Category 1: Models Using "Timing of Food Intake" Results Shiftwork models using shifted and/or restricted timing of food availability are based on the knowledge that food intake is the most important Zeitgeber for peripheral clocks, in the same Barf et al., 2010 Barf et al., , 2012a Barclay et al., 2012; Husse et al., 2012 Food intake total 1/2 1/2 +: 2/4 (50%) −: 0/4 (0%) o: 2/4 (50%) Barf et al., 2010 Barf et al., , 2012a Barclay et al., 2012; Husse et al., 2012 Activity total 0/1 0/2 +: 0/3 (0%) −: 0/3 (0%) o: 3/3 (100%) Barclay et al., 2012; Barf et al., 2012a; Husse et al., 2012 EE total 0/1 +: 0/1 (0%) −: 0/1 (0% Barf et al., 2010 Barf et al., , 2012a Barclay et al., 2012; Husse et al., 2012 Lipid metabolism 2/2 +: 2/2 (100%) −: 1/2 (50%) ∼: 0/2 (0%) o: 0/2 (0%) Barclay et al., 2012; Husse et al., 2012 Corticosterone 1/2 1/2 +: 2/4 (50%) −: 0/4 (0%) ∼: 0/4 (0%) o: 2/4 (50%) Barclay et al., 2012; Barf et al., 2012a,b; Husse et al., 2012 Melatonin Leptin 2/2 2/2 +: 1/4 (25%) −: 3/4 (75%) ∼: 0/4 (0%) o: 0/4 (0%) Barclay et al., 2012; Barf et al., 2012a,b; Husse et al., 2012 Ghrelin BP/heart rate Gene expression 2/2 2/2 (100%) Barclay et al., 2012; Husse et al., 2012 For detailed description of the columns, see Methods section. way as light is for the central clock. Shifting timing of food intake disrupts the orchestrated synchrony between peripheral and brain clocks, which might lead to metabolic problems as peripheral organs such as liver and muscle are essential for energy homeostasis. Shifting the timing of food intake is an interesting approach as metabolic disorders such as obesity are also associated with aberrant dietary habits (i.e., quantity, composition and frequency) and shiftworkers also have changed dietary habits. Moreover, more recently several studies have suggested that also the timing of food intake is crucial to maintain energy homeostasis (Gluck et al., 2011; Garaulet et al., 2013; Hibi et al., 2013; Garaulet and Gomez-Abellan, 2014; Wang et al., 2014) and shifting the timing of food intake is another characteristic feature of shiftworkers (Lowden et al., 2010) . All in all making this a relevant model for shiftwork. Natelson et al., 1993; Dauchy et al., 2010; Fonken et al., 2010; Gale et al., 2011; Coomans et al., 2013a; Aubrecht et al., 2014; Borniger et al., 2014 Food intake total 0/2 2/7 +: 1/9 (11, 1%) −: 1/9 (11, 1%) o: 7/9 (77, 7%) Dauchy et al., 2010; Fonken et al., 2010; Coomans et al., 2013a; Shi et al., 2013; Aubrecht et al., 2014; Borniger et al., 2014 Activity The first evidence for food as a strong entrainment signal for circadian physiology (metabolic and clock gene expression rhythms, hormone secretion rhythms) came from so-called restricted feeding studies. This type of studies usually restricts food availability to a short period (e.g., 2-4 h) during the light phase (which is the resting phase of nocturnal rodents) to study entrainment of peripheral clocks. Clearly these are important studies for chronobiology in general and still are performed frequently to look for and try to understand better the food entrainable oscillator. However, such restricted-feeding models are not Vilaplana et al., 1995; Tsai et al., 2005; Bartol-Munier et al., 2006; Oishi, 2009; Gale et al., 2011; Karatsoreos et al., 2011; Oishi and Itoh, 2013; Voigt et al., 2014 Food intake total 2/3 1/3 +: 2/6 (33, 3%) −: 1/6 (16, 7%) o: 3/6 (50%) Vilaplana et al., 1995; Tsai et al., 2005; Bartol-Munier et al., 2006; Oishi, 2009; Karatsoreos et al., 2011; Oishi and Itoh, 2013 Activity total 2/2 +: 0/2 (0%) Ghrelin BP/Heart rate 1/1 Results Gene expression 1/1 1/1 (100%) Oishi and Itoh, 2013 For detailed description of the columns, see Methods section. an adequate reflection of human food intake behavior during shiftwork as they restrict the duration of food intake to a (very) short period and therefore were not included in this review. Food restriction studies in which food availability is shifted or restricted to a certain phase of the day (i.e., a large part of or the complete light period or dark period) provide a more suitable approach to mimic human feeding behavior during shift work. Results One of the first studies with food availability restricted to either the 12-h light or 12-h dark phase was done by Damiola et al. and they showed a strongly disturbed circadian rhythm according to altered daily body temperature rhythms in mice that could eat only during the light (i.e., resting) phase. Several clock genes in liver adjusted their expression to the timing of food intake (Damiola et al., 2000) . Although alterations in gene expression cannot be translated directly into functional changes, it does indicate that food has strong entraining properties even on a molecular level in mice. More recently, Bray et al. performed a short-term experiment and observed metabolic changes within the first 9 days after restricting food intake to the light or dark phase. Whole body energy metabolism was affected within 24 h of food restriction and this was visible in a 5 h phase advance of rhythm in energy expenditure, higher resting energy expenditure (RER) and increased caloric intake. Restricting food to the resting phase caused an increase of bodyweight and blunted plasma glucose and corticosterone rhythm, whereas triglyceride levels were not affected (Bray et al., 2013) . A short term experiment by Oyama et al. focused on the effects of food timing on inflammatory response but additionally found reduced food intake and bodyweight in mice fed during the light phase (Oyama et al., 2014) . Jang et al. performed the same restriction protocol but studied long-term effects (5-9 weeks). Surprisingly, they observed a protective effect of restricting food to the resting phase with lower bodyweight and food intake when compared to (chow or highfat diet) ad libitum fed animals. Bodyweight did not differ from animals pair-fed during the active phase. Also, alterations in the expression of lipogenic, gluconeogenic and fatty acid oxidationrelated genes in liver were found in feeding time-restricted animals (Jang et al., 2012) . Shamsi et al. entrained mice to 16L:8D or 8L:16D photoperiods and restricted food availability to light-or dark phase. Neither photoperiod nor food timing affected bodyweight when compared to ad libitum feeding. Plasma insulin increased in light phase fed animals despite the photoperiod, whereas plasma glucose tended to be lower and triglycerides significantly decreased when feeding during light was compared to ad libitum or dark phase feeding. Rhythms in plasma glucose, insulin and triglyceride secretion shifted by light phase feeding when compared to dark phase feeding and some effects (mostly amplitude) changed over time (i.e., 7 vs. 35 days). Interestingly, long photoperiod caused light phase fed animals to increase glucose tolerance but decrease insulin tolerance compared to dark phase fed animals and ad libitum fed animals respectively. Gene expression of metabolic and clock genes in liver was altered by feeding during light phase in both amplitude and phase. Corticosterone rhythm was shifted by light phase feeding after 35 days but not after 7 days (Shamsi et al., 2014) . Reznick et al. took a similar approach with a 3-week study performed with Wistar rats instead of mice. No effect was found on bodyweight gain or epididymal white adipose tissue, but animals fed during the resting phase decreased their food intake and total activity levels. Rats fed during the light period showed a 12-h shift in RER and dampening of activity and energy expenditure diurnal variation. The rhythm of plasma insulin altered with higher 24-h levels, corticosterone showed an additional peak and the rhythm of glycogen shifted to an opposite phase in liver but showed increased levels in muscle. Triglyceride levels in liver were reduced whereas muscle content was unaffected in animals fed during the light period. Expression of several other proteins and genes involved in energy metabolism and clock regulation in liver and muscle tissue showed phase changes or altered expression levels. In the same study this experimental design was used for a group of rats fed a high fat diet which aggravated many of the observed effects found in chow day-fed animals with additional disruption of leptin and NEFA (non-esterified fatty acids) levels (Reznick et al., 2013) . Results A series of studies performed with male Wistar rats used a forced activity protocol as a model for shiftwork (SalgadoDelgado et al., 2008 (SalgadoDelgado et al., , 2010a (SalgadoDelgado et al., ,b, 2011 (SalgadoDelgado et al., , 2013 . The effects of forced activity will be described below (see category 2), but the nonworking "control" groups of these studies are relevant for the timing of food category. When food was restricted to the resting phase, i.e., chow only available from ZT0 to ZT12, rats displayed a dampening of their core body temperature rhythm, an additional peak in the plasma corticosterone rhythm, and a shift in triglyceride secretion, but no differences were observed for the plasma glucose and activity rhythm or total activity. Total food intake remained the same but bodyweight and peritoneal fat accumulation were increased when compared to ad libitum or night fed (food available from ZT12 to ZT24) animals (SalgadoDelgado et al., 2010a) . The observed accumulation of abdominal fat was reproduced by the same group in another study where a decreased glucose tolerance in rats fed during the resting phase was observed, in addition to alterations or dampened rhythms in liver clock and metabolic gene expression (Salgado-Delgado et al., 2013) . Results A couple of other groups used comparable food availability approaches in mice on normal chow diet, however, shorter food restriction periods were used than 12 h during the light period. Yoon et al. enforced a 6-h advance (ZT6-11) or delay (ZT18-23) in food availability for 9 weeks and observed that body temperature, locomotor activity and triglyceride secretion strongly depend on food timing. Cholesterol and HDL levels were moderately increased in both advance and delay groups when compared to ad libitum fed animals, and food intake was reduced in the food time advanced group compared to food time delay group and adlib controls. Fasting glucose levels increased and poor responses to insulin tolerance test intensified over time in daytime fed animals (advance group) (Yoon et al., 2012) . Sherman and colleagues restricted food intake to the light phase, ZT4-8 without caloric restriction, for 18 weeks and performed this with both high-and low-fat (chow) diets. In both diets, time restriction was protective for bodyweight gain, high plasma leptin, insulin, HDL and cholesterol levels. Also the increased epididymal fat observed in ad libitum fed animals was diminished in the food time-restricted groups. In low fat diet fed animals triglyceride levels were reduced but corticosterone and adiponectin were increased, as compared to the ad libitum low and high-fat and restricted high-fat animals. High-fat diet fed animals also showed improved TNF-alpha and HOMA-IR levels and increased activity levels when food was restricted to the light period, as compared to ad libitum fed animals, but were less active than animals on a low-fat diet (Sherman et al., 2012) . Schroder et al. focused on the effects on heart rhythm and observed that when food was provided from ZT2-9 only, this negatively affected several aspects of heart rhythm aspects in both wild type and genetically sensitive mice (Schroder et al., 2014) . Results Most studies mentioned above were performed with normal chow diet which is low on fat derived content. However, many diet-induced obesity studies use high-fat diet ad libitum feeding on which animals will develop obesity, diabetes and metabolic syndrome (Zaragoza and Felber, 1970) . For circadian studies it is important to know that feeding rodents a high-fat diet induces a dampening of the amplitude of daily activity and feeding rhythms, and also metabolic markers, hypothalamic neuropeptides and peripherally expressed factors involved in lipid metabolism are affected (Kohsaka et al., 2007) . This suggests an interaction between energy metabolism and circadian rhythm control. Some groups, however, combined the restricted-feeding paradigm with a high-fat diet. Restriction of food to one phase of the day may re-induce the entrainment lost on high-fat ad libitum feeding. Arble and colleagues made an early attempt and fed mice a high-fat diet solely during the light-phase and observed a significant increase in bodyweight compared to animals fed during the dark phase (Arble et al., 2009 ). Bray et al. used four different feeding schedules to study in more detail which phase of the L/D-cycle is most detrimental when ingesting a highfat diet for 12 weeks. They observed higher bodyweight gain and adiposity, decreased glucose tolerance next to high insulin, leptin and triglyceride levels in mice consuming their high-fat meal at the end of the active phase instead of at the beginning. Interestingly, when fat was only available in the light phase no metabolic changes were observed with the exception of slightly decreased energy expenditure and oxygen consumption when compared to animals with fat available in the dark phase (Bray et al., 2010) . Several studies experimented with restriction of a nutrient component to a certain phase of the day for the effects on obesity. For instance, providing the fat component or sugar component of a free-choice high-fat-high-sugar diet only during the light phase affects RER, energy expenditure and bodyweight Oosterman et al., 2014) . A slightly different approach was taken by Senador et al. by offering mice a 10% fructose solution additional to their normal chow and water diet. Fructose was either available for 24 h, available for 12 h during light phase, available for 12 h during dark phase or not available. Increased bodyweight, higher fasting glucose levels but decreased plasma triglycerides were observed in both groups with 12 h fructose availability. Fructose restriction to light phase additionally caused glucose intolerance and an attenuated amplitude of blood pressure rhythms. Ad libitum availability of fructose only caused glucose intolerance when compared to control animals without fructose (Senador et al., 2012) . Another type of timed food restriction is done by dividing food intake into 4 or 6 meals equally divided over the L/D-cycle. For instance, Yamajuku et al. delivered a high cholesterol diet to rats in a 4-meal schedule (every 6 h) without reduction of caloric intake. Those animals developed hypercholesterolemia after 7 days on the protocol and furthermore showed disruption in liver gene expression (Yamajuku et al., 2009 ). These studies are probably highly relevant as shiftwork models. However, until now it is unclear how shift workers exactly change their dietary habits in timing, composition, frequency and size of meals, making it hard to decide at present what are the best models. Results In contrast to studies that restrict food availability to the light phase, Hatori et al. showed that restricting a high-fat diet to the natural main feeding phase (ZT13-21) improved glucose tolerance, insulin sensitivity, adiposity, serum cholesterol levels and leptin levels after fasting or glucose administration, next to prevention of increased liver size and unsaturated fatty acids levels, compared to animals fed ad libitum (Hatori et al., 2012) . Summary "Timing of Food Intake" Models Animal models using a restriction of the timing of food intake affect bodyweight in 64% of the studies, but effects went in different directions with 3 out of 7 studies showing an increase of bodyweight whereas the four other studies found a decrease in bodyweight after timed food intake. Restricting food to the light phase resulted in increased bodyweight compared to animals with food restricted to the dark phase (Arble et al., 2009; Bray et al., 2013) and when compared to ad libitum fed (SalgadoDelgado et al., 2010a) . However, others observed reduced bodyweight after food restriction to the light phase compared to dark fed animals (Yoon et al., 2012; Oyama et al., 2014) or ad libitum fed animals (Sherman et al., 2012; Yoon et al., 2012) . Interestingly, in many of these studies food intake was reduced as well. Furthermore, some differences between mouse and rat studies are observed. For example, for bodyweight, mice studies show a significant effect in 6 out of 8 studies (75%), whereas in rats only in 1 out of the 3 studies (33%) a significant effect was observed. Total food intake and glucose metabolism parameters were measured frequently and were affected in 45 and 67% of the studies, respectively. For a complete overview of all parameters see Table 1 . Together, these results indicate that changing the timing of food intake is effective at influencing several metabolic parameters, although for some parameters results are not consistent. Considering the great variety in types of modulations used it is not possible to pinpoint this to one aspect of the models used. Summary "Timing of Food Intake" Models Category 2: Models Using "Timing of Activity" Summary "Timing of Food Intake" Models "Work" is a difficult concept to model in animal studies and therefore only a very limited number of studies are available using an actual physical shiftwork protocol. Obviously shiftwork requires shifting of phases of sleep and arousal leading, at least partly, to awakening during the usual sleep period and sleep during the usual active period of the day. The few available physical shiftwork models used forced activity by housing animals in slowly rotating wheels. These housing conditions force the animals to be active and prevent them to fall asleep, although the animals can lie down and eat. Salgado-Delgado et al. mimicked human shiftwork protocols by enforcing the working conditions for 8 h per day, either during the active phase or the sleeping phase, 5 days a week for 5 weeks. In addition, they varied the availability of food but always used normal chow diets. A general observation in the groups of animals working during their resting phase was that the animals lost their nocturnal urge to eat and voluntarily consumed their food mostly during their working hours and thereby during their normal resting (i.e., light) phase. Forced activity during the resting phase induced increased bodyweight and abdominal fat, impaired glucose tolerance, altered plasma triglyceride diurnal variation, dampened daily glucose variation and introduced a secondary peak in the corticosterone rhythm. These effects could be prevented when food availability was restricted to the normal active (i.e., dark) phase (Salgado-Delgado et al., 2008 , 2010a , 2013 ). Summary "Timing of Food Intake" Models An early study by Tsai et al., used an extensive design with rats exposed to changes of light schedules (twice a week), forced activity paradigms and combinations of those. They observed higher bodyweight in the first 2 months, but not in the third month, in animals that were subjected to a "shiftwork" schedule of forced activity (i.e., Tue-Thu work from ZT0-ZT12, Fri-Sun work from ZT12-ZT24, Monday free) combined with changes in light schedule which was in synch (i.e., 12 h shift of L:D cycle twice a week) with the work schedule. Animals exposed to forced activity schedules had lower bodyweight and lower levels of cholesterol than animals that only underwent shift of light/dark cycle, indicating that forced activity can reverse some effects on metabolism induced by the regular L/D shifts. Some early effects on bodyweight and food intake disappeared after 2 months on the protocol, whereas other effects were only found after a few months on this protocol (Tsai and Tsai, 2007) . Summary "Timing of Food Intake" Models A study by Leenaars et al. using forced activity in rats did not focus on metabolic parameters, but did observe a decrease in total activity levels in animals that had to work either during their active or during their resting phase compared to freely active animals. Animals working shiftwork (i.e., work during the normal resting phase) showed reduced bodyweight gain compared to non-working controls (Leenaars et al., 2012) . Summary "Timing of Food Intake" Models A study by Hsieh et al. used 5-week forced activity in rats and closely studied alterations in activity patterns. The animals in this study are from the same study as Salgado-Delgado et al. (2008) but reported more specifically on locomotor activity. A decrease in mean activity levels was observed in shiftwork animals (i.e., working ZT2-10) during weekdays from week 3 onwards and during weekend days from week 1 onwards, but not in animals working during their active phase (i.e., working from ZT14-22). Shiftwork animals showed decreased amplitude of the activity rhythm on weekend days and a differently shaped rhythm of 24-h activity on weekdays. Shiftwork animals showed different activity responses to lights on and off when compared to nonshiftworking rats (Hsieh et al., 2014) . The observed disruption of normal activity patterns was reported as an indication for circadian disruption as similar changes have been observed in SCN lesioned animals. Summary "Timing of Activity" Models Models using an altered timing of activity have only been carried out with rats and therefore no numbers on mouse studies are available. The limited number of studies and contributing research groups with this paradigm resulted in high numbers of affected studies on all parameters. As shown in Table 2 , all parameters showed a 100% effectiveness of the different studies, except for bodyweight, total food and total activity levels intake which were affected in 5, 2, and 4 out of 6 studies, respectively. These results suggest that "timing of activity" has metabolic effects, although considering the limited number of studies and research groups these results should be interpreted with some caution. Category 3: Models Using "Timing of Sleep" Alterations in timing of activity are directly related to alterations in timing and duration of sleep. However, changes in the timing of sleep are also separately used as a model for shiftwork. These studies are different from the previously described activity models as their first target is to disturb or shorten sleep and affect the timing of sleep, but not necessarily alter activity levels or food intake. However, undoubtedly changes in sleep behavior will also affect activity and feeding patterns. Interestingly, (chronic) sleep restriction is associated with metabolic disorders in both animals and humans (Gangwisch, 2009; Killick et al., 2012) . We came across a diversity of methods used to disrupt the normal sleep/wake pattern, including sleep disruption, sleep restriction, sleep fragmentation, sleep perturbation or sleep deprivation. Some of those might be other designations of the same intentions, such as sleep fragmentation and perturbation. These models either use shifting the timing of the normal sleep phase along the light-dark cycle, perturbing sleep in the normal phase, reducing the total number of sleep hours or completely withholding sleep. Most of these sleep studies focused on the effects of sleep perturbation on sleep parameters (such as percentage REM and NREM sleep, EEG recordings), behavioral changes or other non-metabolic factors. In this review only studies using a shift in the timing of sleep to the dark phase and studies using total sleep deprivation for a few hours were included as those were considered most relevant for shiftwork models. Category 3: Models Using "Timing of Sleep" Methods to perturb sleep are diverse and forced activity, as mentioned before, is one of them. Another method involves gentle handling for a few hours, by touching the animal every time it tries to fall asleep. Short term effects of sleep restriction during the first 6 h of the normal sleep phase (ZT0-ZT6) were described in two studies. Barclay et al. observed moderate alterations in the timing of food intake (in the direction of light phase feeding) and locomotor activity (increased levels during light and decreased levels during dark phase), next to major disruptions in liver transcriptome rhythms enriched for lipid and glucose metabolism pathways after 2 weeks of sleep restriction. A decreased response in the pyruvate test, a dampening of the daily rhythms in plasma glycerol, plasma triglyceride, plasma corticosterone and hepatic glycogen levels, and disrupted expression of several clock genes were all rescued by restricting food intake to the dark phase in the sleep restricted groups (Barclay et al., 2012) . Category 3: Models Using "Timing of Sleep" Another study used the same method but subjected the animals to sleep restriction for only five consecutive days followed by a recovery week during which several parameters were measured. Sleep restriction led to increased food intake despite increased leptin levels, together these changes are indicative for leptin resistance. Blood metabolites such as glucose and triglycerides were increased, but levels improved again during the recovery week. However, a trend toward higher bodyweight gain was observed during the recovery week suggesting long term effects even when the period of sleep restriction has terminated. Analysis of white adipose tissue transcriptome showed that sleep restriction affects many genes involved in lipid metabolism, including increased fatty acid synthesis and triglyceride production and storage. Category 3: Models Using "Timing of Sleep" A series of sleep restriction studies (Barf et al., 2010 (Barf et al., , 2012a ,b) have been done using sleep restriction protocols of different severities: sleep restriction for 20 h each day (sleep ZT0-4; awake ZT4-24) or sleep disruption (14 h sleep-10 h awake in four 2-3 h episodes). Sleep disturbance and sleep restriction both led to decreased bodyweight when compared to home cage control animals, although food intake was equal and the slight increase in locomotor activity is unlikely to explain the bodyweight differences. Animals exposed to sleep disruption or sleep restriction showed decreased baseline plasma glucose and insulin levels, decreased glucose tolerance and an attenuated insulin response to the glucose infusion within 8 days. A five day recovery period attenuated the sleep restriction-induced decrease of plasma leptin, insulin and glucose levels although bodyweight gain did not recover. Corticosterone levels and food intake were not affected (Barf et al., 2012b) . When the same protocol was performed for 4 weeks, but with a work-weekend-schedule (5 work days, 2 nonwork days) to resemble human shiftwork conditions, the same authors observed an increase of food intake after the first weekend, possibly to compensate for increased energy expenditure. Bodyweight gain increased during weekends in sleep restricted animals and plasma leptin and insulin levels were decreased when measured during working weeks 1 and 4. Rest during weekend days induced recovery of plasma leptin and insulin levels (Barf et al., 2012a) . Summary "Timing of Sleep" Models The number of studies using perturbation of sleep as a model for shiftwork with focus on metabolic parameters is limited and therefore it is not yet completely clear if this type of manipulation influences metabolic functioning. Glucose metabolism appears to be affected often (in 5 out of 5 studies), whereas bodyweight (3/5) and food intake (2/4) were not always affected by sleep perturbation. For a complete overview of all parameters see Table 3 . Summary "Timing of Sleep" Models Category 4: Models Using "Timing of Light Exposure" Summary "Timing of Sleep" Models In literature, several "timing of light exposure" models have been reported. These models all use timing of light as a means to disturb the circadian system and as such their main influence is on the SCN, in contrast to the previous 3 categories of models described. One type of these models uses continuous light exposure, i.e., light is present 24 h per day. The continuous light models can be further subdivided in models using constant light (similar amounts of light exposure during 24 h of the day) and models using dim light at night (together with bright light during the day). In addition to models using continuous light, models using changes in light/dark schedules have been used. These models can be further subdivided into models using alterations in period length (e.g., light-dark periods shorter or longer than 24 h) and models using repeated shifts of the light/dark schedule. Studies investigating these types of exposure in relation to metabolic health effects are discussed below per type of model. For overview purposes, models using continuous light are presented together in Table 4 and models using changes in light/dark schedules are presented together in Table 5 . Continuous Light Exposure Exposure to constant bright light conditions (LL) has been shown to abolish/diminish the circadian rhythmicity of many laboratory animals. This has, for example, been shown for locomotor activity (Depres-Brummer et al., 1995; Gale et al., 2011) , melatonin (Wideman and Murphy, 2009; Gale et al., 2011) , food intake (Coomans et al., 2013b) , and SCN neurons (Coomans et al., 2013b) . Hence, constant bright light conditions may be used to severely disrupt circadian rhythms. Since shiftworkers will be exposed to light during most of the day these models might be relevant for shiftwork modeling, although the intensity of all-day light may differ from real-life light exposures. Continuous Light Exposure Disruption of circadian rhythms by constant bright light exposure has also been reported to affect metabolic function. Exposure to continuous light (150-180 lux; 4-8 weeks) led to increased bodyweight gain in two studies in mice (Fonken et al., 2010; Coomans et al., 2013a) . This phenotype was apparent in mice fed normal chow (Fonken et al., 2010; Coomans et al., 2013a) as well as a high-fat diet (Coomans et al., 2013a) . In addition, a third study reported increased fat mass after exposure to continuous light (Shi et al., 2013) . Total food intake was unaltered in these studies, but more food was consumed during the subjective day, indicating changes in the timing of food intake (Fonken et al., 2010; Coomans et al., 2013a) . The effect of continuous light exposure on total activity levels is less clear: unaltered activity levels (Fonken et al., 2010) , reduced energy expenditure (Coomans et al., 2013a ) and a non-significant trend toward a decrease in activity levels (Shi et al., 2013) have been reported. Apart from changes in bodyweight and fat mass, continuous light affected other metabolic parameters, such as increased RER, reduced glucose tolerance and altered rhythmicity of insulin sensitivity (Fonken et al., 2010; Coomans et al., 2013a) . However, two studies using relative short-term exposure (6-10 weeks) to continuous bright light in Sprague Dawley rats reported no changes in bodyweight (Dauchy et al., 2010; Gale et al., 2011) , indicating a possible difference between rats and mice in this model. Interestingly, long term exposure to bright light for 35 weeks did enhance bodyweight in Rapp-Dahl rats (model for hypertension). In addition, increased systolic blood pressure was observed (Natelson et al., 1993) . Short-term exposure to continuous bright light did increase glucose levels and alter the rhythmicity of lipids in the study by Dauchy et al. (2010) . In contrast, in the study by Gale et al. changes in glucose metabolism (increased glucose levels and decreased glucose-and arginine-stimulated insulin secretion) were only observed in diabetes-prone HIP rats, but not in wild-type Sprague Dawley rats (Gale et al., 2011) . Considering these contradicting results, the differences in duration and strains of rats used, as well as the limited number of studies no firm conclusions can be drawn regarding the effects of continuous bright light exposure on metabolic function in rats. On the other hand, in mice results are more consistent and indicate that disruption of circadian rhythms by continuous bright light exposure increases bodyweight and alters glucose metabolism. This is associated with an altered timing of food intake, but not with change in total amount of food consumed over 24 h ( Table 4) . Dim Light at Night Dim light at night (LDim) also affects circadian rhythmicity, but seems less disruptive for circadian rhythms compared to constant bright light. In respect to human circadian disruptions caused by shiftwork, models using dim light might be more relevant, since human shiftworkers will also experience alterations in the level of light exposure during a day, i.e., dim light at work in the office and bright light when commuting. In addition, models using dim light at night are relevant for studying the possible health consequences of light contamination at home, i.e., evening and nocturnal light is present in an increasing amount in our western society. Dim Light at Night In contrast to constant bright light exposure, with dim light exposure at night
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