Intermittent fasting (IF) can improve function and health during aging in laboratory model organisms, but the mechanisms at work await elucidation. We subjected fruit flies (Drosophila melanogaster) to varying degrees of IF and found that just one month of a 2-day fed:5-day fasted IF regime at the beginning of adulthood was sufficient to extend lifespan. This long-lasting, beneficial effect of early IF was not due to reduced fecundity. Starvation resistance and resistance to oxidative and xenobiotic stress were increased after IF. Early-life IF also led to higher lipid content in 60-day-old flies, a potential explanation for increased longevity. Guts of flies 40 days post-IF showed a significant reduction in age-related pathologies and improved gut barrier function. Improved gut health was also associated with reduced relative bacterial abundance. Early IF thus induced profound long-term changes. Pharmacological and genetic epistasis analysis showed that IF acted independently of the TOR pathway because rapamycin and IF acted additively to extend lifespan, and global expression of a constitutively active S6K did not attenuate the IF-induced lifespan extension. We conclude that short-term IF during early life can induce long-lasting beneficial effects, with robust increase in lifespan in a TOR-independent manner, probably at least in part by preserving gut health.

Here, we investigated a variety of IF regimes in flies and their effects on a range of health outcomes, including feeding behavior, gut and metabolic health, survival after stress, and lifespan. Importantly, short-term IF (the “2:5” diet) confined to early life robustly increased subsequent lifespan, particularly in females, independent of TOR signaling. Short-term IF also led to long-lasting health improvements, including increased stress resistance and a lower incidence of gut pathology that was associated with reduced bacterial abundance.

Previous studies examining potential pro-longevity effects of IF in flies have produced mainly negative results []. The first studies, almost 90 years ago, found that 6 hr of starvation in every 24 hr was beneficial and could extend lifespan []. However, the effects of this IF regime may be strain or food medium specific, because a similar, more rigorous experiment ∼80 years later found that daily bouts of either 3 hr or 6 hr starvation throughout the adult life of the fly had neither a positive nor a negative effect on lifespan []. Interestingly, short-term fasting can increase resistance to severe cold stress [] and facilitate long-term memory formation in flies []. Time-restricted feeding (TRF) in Drosophila improves sleep consolidation as well as a variety of cardiac output functions that normally decline with age, despite the caloric ingestion/expenditure of TRF flies being the same as ad-libitum-fed flies []. Thus, health improvements can result from various IF regimes in Drosophila, but the mechanisms at work await elucidation and the evidence for a pro-longevity phenotype from IF is more mixed.

On the influence of intermittent starvation on the longevity of the imaginal stage of Drosophila melanogaster.

On the influence of intermittent starvation on the longevity of the imaginal stage of Drosophila melanogaster.

Invertebrate model organisms provide powerful contexts in which to establish the molecular mechanisms mediating the pro-longevity effects of IF. In yeast, Rim15, a key integrator of signals transduced by the Sch9, Ras, and TOR pathways, is important for starvation-induced lifespan extension [] while in the nematode worm Rheb-1, with a key role in TOR signaling, is essential for the pro-longevity effects of IF []. Interestingly, the findings with both organisms imply the existence of additional underlying mechanisms mediating the pro-longevity effects of IF. It is unclear whether Rim15/Rheb-1 and their interactors mediate the effects of IF in mammals.

Reduced activity of nutrient-sensing pathways, with corresponding decrease in global protein translation, is implicated as an important mechanism underlying the pro-longevity effects of dietary interventions, such as DR []. Reduced TOR signaling is a hallmark of pro-longevity interventions, including DR, and treatment with the TOR inhibitor rapamycin extends healthy lifespan in a range of organisms []. Although DR may exert some of its pro-longevity effects through reduced fecundity, DR can still extend lifespan in sterile, ovomutant Drosophila, implying that fecundity and lifespan can be uncoupled and that other mechanisms are also important []. A recent study highlighted the importance of gut homeostasis in DR-induced longevity in Drosophila, because DR both rescued age-related gut pathologies and extended lifespan in females and in males with feminized guts, but not in wild-type males, which do not undergo significant age-related gut pathology []. Diet composition and fly food transfer schedule are also known to affect the abundance and diversity of fly-associated bacteria [], and periods of fasting may be expected to modulate the associated microbiota.

DR, a chronic reduction of food intake without malnutrition, is an evolutionarily conserved method of improving health during aging and extending lifespan []. However, many studies of DR, particularly in rodents, also involve intermittent access to food, with the DR animals gorging their reduced meal as soon as it is supplied, leaving extended periods of time in a fasted state []. The beneficial health effects seen in DR may therefore be attributable, at least in part, to intermittent starvation. Supporting this idea, performing DR without the extended fasting periods by diluting the food of the DR mice with non-digestible cellulose, thus restricting total energy intake but allowing constant access to food, did not extend lifespan compared to fully fed animals []. In contrast, many studies in invertebrate organisms, including yeast, worms, and Drosophila, where the DR treatment involves continuous access to diluted food, result in robust lifespan extension []. Therefore, periodic fasting may be important for, but is likely not the only contributor to, the pro-longevity effects of DR, at least in these invertebrates.

IF can extend lifespan in a variety of organisms, including bacteria, yeast, nematode worms, and mice []. In animal models, IF has been shown to reduce the risk of developing a variety of age-related pathologies []. IF is effective in preventing neurodegeneration in rodents [] and can attenuate cancer [] and cardiometabolic diseases, such as type II diabetes []. FMD was recently found to increase pancreatic β cell regeneration in mouse models of diabetes [].

Intermittent fasting (IF), an umbrella term for diets that cycle between a period of fasting and non-fasting, has become increasingly popular as a weight loss regime (e.g., “every-other-day fasting” and the “5:2” diet) []. Advocates of IF argue that it shows many of the benefits seen with traditional daily energy restriction diets but with a simplified nutritional regime and increased compliance []. One study on the clinical outcomes of fasting in young overweight women described significant weight loss as a result of the IF regime, as well as reduced fat mass and waist circumference, and lowered serum cholesterol, triglycerides, and C-reactive protein []. More recent, pilot, clinical trials used a fasting mimicking diet (FMD) (consisting of monthly cycles of a 5-day fast during which daily food intake was reduced to ∼50% normal caloric intake), which reduced multiple health risk factors during the post-fast recovery period, including lowered blood pressure, and reduced blood glucose and insulin-like growth factor-1 (IGF-1) levels []. However, systematic reviews of the clinical benefits of fasting regimens in humans found that study designs were heterogeneous and compliance data limited, making it difficult to draw definitive conclusions [].

To further elucidate the mechanisms mediating the lifespan-extending effects of IF, we also performed genetic epistasis experiments. S6K facilitates the downstream effects of TOR by its effects on protein translation, and rapamycin-induced lifespan extension was previously shown to be abrogated by its upregulation []. We therefore examined the effects of IF on flies ubiquitously expressing a constitutively active form of S6K (UAS-S6K) []. In two independent experiments, we found that IF led to significant lifespan extension in Da-GAL4 driver-alone heterozygotes compared to ad libitum controls, whereas ubiquitous overexpression of the constitutively active S6K did not abolish the lifespan-extending effects of IF ( Figure 4 C). Cox proportional hazards analysis of the pooled lifespan data found no significant interaction between IF and S6K. We can therefore conclude that IF is not epistatic with, and is acting independently of, TOR.

We first examined the effect of administering rapamycin after 30 days of the IF regime and compared these with IF vehicle (EtOH) and IF controls ( Figure 4 A). We found that the lifespan-extending effects of rapamycin and IF were additive, indicating distinct mechanisms. Using a different experimental setup, instead of administering rapamycin after the early-life IF period, we administered rapamycin to flies throughout the entire lifespan ( Figure 4 B). Compared to untreated ad libitum controls, ad libitum rapamycin and IF switch both extended lifespan to a similar extent. During the IF regime, flies have access to food for 2 days per week, so to control for feeding fasted flies rapamycin during the 2-day “fed” period, we also added an ad libitum condition where we fed rapamycin only during the 2-day fed period. We observed a small but significant lifespan extension in flies fed rapamycin 2 days per week compared to untreated ad libitum controls. Rapamycin was protective against the deleterious effects of lifelong IF. Treatment with rapamycin during the 2 day fed period of the IF switch diet then ad libitum thereafter resulted in increased lifespan compared to the untreated IF switch condition. Similarly, for flies treated with rapamycin during the 2-day fed period of the IF switch diet then ad libitum rapamycin thereafter, lifespan was increased compared to the untreated IF switch condition. Compared to the already extended lifespans of IF switch or rapamycin treatments alone, flies that were exposed to both IF and rapamycin, whether with ad libitum food or ad libitum rapamycin during the post-IF period, further extended lifespan to a similar extent. Cox proportional hazards analysis of the lifespan found no significant interaction between IF and rapamycin, indicating IF and rapamycin have independent effects on lifespan. Therefore, the lifespan-enhancing effects of IF and rapamycin are additive, indicating non-overlapping mechanisms.

(C) Two replicate lifespan experiments examining the effect of constitutive S6K activation on the pro-longevity effect of IF. IF led to significant lifespan extension in Da-GAL4 driver-alone heterozygotes compared to ad libitum controls (left panel, p = 8.7 × 10 −05 ; right panel, p = 7.7 × 10 −21 ; log rank test). Ubiquitous overexpression of the constitutively active S6K (UAS-S6K STDETE ) via Da-GAL4 did not abolish the lifespan-extending effects of IF (left panel, p = 4.0 × 10 −12 ; right panel, p = 0.0027; log rank test). There was no significant interaction between IF and S6K (p = 0.10), indicating IF and S6K had independent effects on lifespan. n > 200 flies per condition for both experiments.

(B) Ad libitum rapamycin treatment (black dashed line) extended lifespan compared to untreated controls (solid black line; p = 0.0003, log rank test). Rapamycin treatment during the “fed” period of the IF diet (blue dashed line) rescued the lifespan-shortening effects of the IF diet for the duration of the lifespan (red dashed line; p = 1.2 × 10 −07 ; log rank test). IF switch (solid red line) extended lifespan compared to ad libitum controls (solid black line; p = 0.00018; log rank test). Rapamycin treatment during the fed period (i.e., 2 days per week) of the IF switch diet and then ad libitum sugar/yeast/agar (SYA) thereafter (solid blue line) led to a further significant increase in lifespan compared to the untreated IF switch condition (solid red line; p = 0.00078; log rank test). Rapamycin treatment for 2 days per week and ad libitum SYA the rest of the time (solid green line) led to a small but significant lifespan extension compared to untreated ad libitum controls (solid black line; p = 0.034; log rank test). Rapamycin treatment during the fed period (i.e., 2 days per week) of the IF switch diet and then ad libitum SYA thereafter with rapamycin treatment 2 days per week (green dashed line) led to a further significant increase in lifespan compared to the untreated IF switch condition (solid red line; p = 6.7 × 10 −05 ; log rank test). Cox proportional hazards analysis of the lifespan demonstrated a significantly reduced risk of dying when flies were exposed to IF versus ad libitum (p = 6.95 × 10 −05 ) or rapamycin versus ad libitum control food (p = 0.00055). There was no significant interaction between IF and rapamycin (p = 0.94), indicating IF and rapamycin had independent effects on lifespan. n > 143 flies per condition.

(A) IF switch and IF switch with EtOH (vehicle control, beginning after IF) extended lifespan compared to ad libitum controls (IF switch, p = 1.4 × 10 −13 , log rank test; IF switch + EtOH, p = 5.0 × 10 −13 , log rank test). Rapamycin treatment after IF (i.e., starting at 30 days) further extended lifespan compared to IF switch and IF switch + EtOH controls (Rapa versus IF switch, p = 0.0081; Rapa versus IF switch + EtOH, p = 0.013 log rank test). n > 200 flies per condition.

We hypothesized that the beneficial effects of IF on lifespan were acting, at least in part, via the TOR pathway. IF appeared to phenocopy many of the beneficial effects on lifespan, stress resistance, and gut health observed with reduced TOR signaling []. Indeed, reduced TOR signaling has been shown to prolong longevity in a variety of organisms []. Additionally, rapamycin can extend Drosophila lifespan beyond the maximum achieved by DR [], indicating that the effects of rapamycin are partially independent of those of DR. Because the effects of rapamycin and DR on lifespan are additive, we examined whether IF and rapamycin were also additive.

The 2:5 diet thus protected against the age-related deterioration of female gut homeostasis, as measured by reduced ISC activity without major ultrastructural changes, and, with reduced tumor-like pathologies in both R2/R4 regions, protected against the loss of gut barrier function and led to reduced bacterial load.

Gut barrier integrity is linked to microbial dysbiosis with age [], and removal of bacteria in late adulthood extends lifespan []. Diet and fly food transfer schedule are also known to affect the diversity and abundance of fly-associated bacteria, and the fasting regime we perform may be expected to modulate the associated microbiota. We therefore compared relative bacterial abundance in 40-day-old (i.e., day 10 post-IF) whole females by qPCR of the bacterial 16S rRNA gene with pan bacterial and Lactobacillus-plantarum-specific primers. L. plantarum was chosen as it is one of the most abundant commensals in the adult fly, and it was also recently associated with loss of gut epithelial integrity in adults []. Relative bacterial abundance was significantly reduced in IF switch flies compared to ad libitum controls using both pan bacterial and L. plantarum primers ( Figure 3 I).

As a functional readout of gut homeostasis, we measured gut barrier function using the smurf assay []. In young flies, FD&C blue dye no. 1 does not normally pass through the gut into the body cavity, but in a small population of old flies, the blue dye can pass through the gut barrier and cause the flies to turn blue (or “smurf”). We found that there were significantly fewer 60-day-old IF switch flies that smurfed compared to ad libitum controls ( Figure 3 H), indicating improved gut barrier function due to IF.

We next examined gut pathology in the R2 and R4 regions of the midgut from 70-day-old females and compared ad libitum, 5-day IF, and IF switch conditions. In R2/R4 regions of young female guts, nuclei are organized typically in a single-layer epithelium, whereas in aged females, the epithelium can exhibit several layers of nuclei, indicating widespread disruption and pathology []. Pathologies were scored blind, binned into scaled categories, and quantified. 70-day-old ad libitum females exhibited several layers of nuclei in both R2 and R4 midgut epithelia, indicating widespread pathology, whereas fully fasted 5-day IF females had reduced pathology, particularly in the R2 midgut region ( Figures 3 E–3G). Similar to ISC activity, IF switch flies had noticeably reduced R2 and R4 midgut pathology compared to ad libitum controls. This confirms the protective effect of short-term early-life IF on age-related tumor-like gut pathologies in females.

We examined the R4 midgut region from 70-day-old females and compared ad libitum, 5-day IF, and IF switch conditions. Mitotically active ISCs were visualized by phospho-histone H3 (pH3) immunostaining. Ad-libitum-fed females exhibited widespread ISC activity, whereas fully fasted 5-day IF females had significantly fewer pH3-positive cells ( Figures 3 A and 3B ), indicating ISC quiescence. Notably, after 40 days since the switch to ad libitum food, IF switch flies still had significantly fewer pH3-positive cells compared to ad libitum controls. Gut length was significantly shorter in fully fasted 5-day IF females compared to ad libitum controls ( Figure 3 C). IF switch guts, however, were not shorter than those of ad libitum controls. We also measured the widths of R4 regions from each gut ( Figure 3 D) and found that, after 5 days of fasting, 70-day-old 5-day IF guts were significantly narrower than ad libitum controls. IF switch guts were not narrower than ad libitum controls. Therefore, the age-related pathology of increased stem cell activity in old female guts, as measured by pH3 staining, was rescued by short-term early-life IF intervention. This protection appeared to be long lasting and was not due to major morphological or structural changes in the gut.

(I) Quantification of relative bacterial abundance in whole females by qPCR of the bacterial 16S rRNA gene with pan-bacterial primers and Lactobacillus-plantarum-specific primers. IF switch flies at day 40 (i.e., day 10 post-IF) had significantly reduced bacterial abundance compared to ad libitum controls (pan bacterial, p = 0.00012; L. plantarum, p = 0.0004; Student’s t test). Values were normalized to the Drosophila GAPDH gene. Data are shown as mean ± SEM (n = 8 biological replicates per condition).

(H) Barrier function at day 60, as measured by “smurf” flies with leaky intestines, was significantly improved in IF switch females compared to ad libitum controls (p = 0.0013; Fisher’s exact; n numbers on panel).

(F and G) Pathologies were binned into scaled categories and quantified. R2 (F, n = 10–12 per condition) and R4 (G, n = 7–10 per condition) categories were defined as follows: I, wild-type (WT), nuclei form single layer epithelium; II, sporadic pathology of small nuclei “nests”; III, intermediate pathology, sections of epithelium have several layers of nuclei; and IV, widespread pathology, majority of epithelium has several layers of nuclei.

(E) Midgut pathology. Representative confocal fluorescence sections of R2 (top row) and R4 (bottom row) midgut regions from 70-day-old ad libitum, 5-day IF, and IF switch flies stained with DAPI (white) to visualize nuclei and anti-βPS-integrin antibody (βPS, green) to visualize the basement membrane/muscle are shown.

(D) Quantification of gut width at R4 section. After 5 days of fasting, 70-day-old 5-day IF guts were significantly narrower than ad libitum controls (p = 0.025; one-way ANOVA). IF switch guts were not significantly different compared to ad libitum controls. 16–18 guts per condition are shown. Data are shown as mean ± SEM.

(C) Quantification of gut length. After 5 days of fasting, 70-day-old 5-day IF guts were significantly shorter than ad libitum controls (p = 0.038; one-way ANOVA). IF switch guts were not significantly different from ad libitum controls. 11–13 guts per condition are shown. Data are shown as mean ± SEM.

(B) Quantification of pH3-positive (pH3+) cells per gut. After 5 days of fasting, 70-day-old 5-day IF guts had significantly fewer pH3+ cells compared to ad libitum controls (p < 0.0001; one-way ANOVA). IF switch guts, after 40 days on food, also had significantly fewer pH3+ cells compared to ad libitum controls (p < 0.0001; one-way ANOVA). 19 or 20 guts per condition are shown. Data are shown as mean ± SEM.

(A) ISC activity. Representative confocal fluorescence z projections of midgut regions 4/5 from 70-day-old ad libitum, 5-day IF, and IF switch flies stained with DAPI (magenta) and anti-phospho-H3 antibody (pH3, green) are shown. The scale bar represents 100 μm.

Maintenance of gut homeostasis has been shown to play an important function in the determination of lifespan in Drosophila []. Midgut tissue homeostasis is maintained by intestinal stem cells (ISCs), which are arranged along the basement membrane and respond to stress and lead to a regenerative response. In the aging female fly gut, ISC proliferation becomes dysregulated and can lead to mis-differentiation and hyperplasia in the intestine. DR reduces gut pathology in aging females [], and we therefore sought to examine the effects of IF on gut homeostasis.

Whole-fly protein levels were not significantly different between any of the conditions tested ( Figures S4 F–S4H). Therefore, the reduction in TAG levels during the 5-day IF period recovered and stabilized to ad libitum levels post-IF. Indeed, it appears that TAG was accumulating in 5-day IF switch flies. Increased TAG levels at 60 days may indicate a mechanism behind the long life of IF switch flies.

Fasting triggers mobilization of internal fat stores, which can enable animals to survive during extended periods of starvation. We therefore performed a time course experiment measuring whole-fly triacylglyceride (TAG) from 5-day IF switch flies. We also examined flies that remained on the 5-day IF regime. Under ad libitum conditions, TAG levels increased with age and plateaued around day 37 ( Figure S4 E). In contrast, 5-day IF flies had significantly reduced TAG levels when fully fasted. On day 37, TAG levels in IF switch flies had not fully recovered to ad libitum levels. However, TAG levels in IF switch flies recovered to ad libitum levels from day 46 (i.e., 2 weeks after the switch to ad libitum food). We examined TAG levels in 60-day-old females (i.e., 30 days since the end of IF diet). Whole-fly TAG levels were significantly higher in IF switch flies compared to ad libitum controls ( Figure S4 G). TAG levels at day 60 from 3-day, 4-day, and 5-day IF switch flies were also examined. TAG levels were significantly higher in 5-day IF switch flies alone compared to the other conditions ( Figure S4 H), highlighting a likely explanation for the greater pro-longevity effect of 5-day IF switch compared to 3- or 4-day IF switch.

We also examined post-IF feeding behavior using the CAFE assay and began measuring at 6 days after the IF switch. Cumulative food intake was significantly higher in IF flies compared to ad libitum controls ( Figure S4 D). We conclude that cumulative food intake was not significantly reduced during IF, whereas feeding was increased after IF. Therefore the IF-induced lifespan extension was not due to a DR-like reduction in total feeding post-IF.

Flies exposed to the 5-day IF regime are fed for 2 days per week. We hypothesized the subsequent lifespan extension observed in IF switch flies could be due to an overall lifelong reduction in calorie intake, as in DR. We therefore examined the effect IF had on total food intake by performing the quantitative capillary feeder (CAFE) assay ( Figures S4 A–S4C). During the 2-day feeding period, 5-day IF flies ingested significantly more food than controls ( Figure S4 A). However, after 14 days, there was no significant difference in cumulative food ingestion between IF flies and controls ( Figures S4 B and S4C).

Cumulative Food Intake Is Not Reduced during IF and Increases after the IF Switch

Most pro-longevity interventions in flies are accompanied by increases in stress responses [], including starvation resistance. We performed a starvation assay on 42-day-old post-IF females ( Figure S3 H) and found that flies previously exposed to IF lived significantly longer. We next examined the effects of dichlorodiphenyltrichloroethane (DDT), a xenobiotic toxin, and paraquat, an oxidative stress, on the survival of 42-day-old post-IF females ( Figures S3 I and S3J). From two independent replicate studies, post-IF flies exhibited a small but significantly enhanced resistance to DDT and paraquat. Therefore, early IF can lead to long-lasting stress resistance. Post-IF flies that were fed DDT and paraquat exhibited increased resistance to these toxins, and we hypothesized that the protective effects of IF may be due, at least in part, to improvements in gut health. In contrast, although DR is associated with increased TAG and starvation resistance [], DR does not increase xenobiotic resistance in flies, and thus IF is distinct from DR in this respect [].

To assess the effects of IF at stages other than during early adulthood, we also examined the effect of a “mid-life” and “late-life” IF on lifespan. We again observed significant lifespan extension in 5-day IF switch flies after 30 days of the regime in early adulthood ( Figure 2 G). To perform a mid-life IF switch, flies were kept on ad libitum food for the first 20 days of adulthood, moved to 5-day IF from days 20 to 45 (25 days of the regime), and then placed back on ad libitum food for the remainder of adult life. We observed a significant lifespan extension in mid-life IF switch flies compared to ad libitum controls, although there was notable mortality before the switch back to ad libitum food. We next assessed the effect of late-life IF on lifespan. Switching wflies to 5-day IF after 30 days of ad libitum food significantly shortened lifespan ( Figure S3 G), indicating the pro-longevity effects of IF are exerted during early life and, to a lesser extent, mid-life.

Every-other-day (EOD) fasting involves alternating feeding/fasting on consecutive days and is less severe than 5 days of continuous fasting. We performed EOD fasting for the first 30 days of life (EOD switch) and found no effect on lifespan compared to ad libitum controls, whereas the 5-day IF switch significantly increased lifespan ( Figure 1 G). We next investigated the optimum duration of early-life IF for extension of lifespan in female flies. Neither 10 nor 20 days of 5-day IF extended lifespan ( Figures 2 A and 2B ), whereas 30 days ( Figure 2 C), 40 days ( Figure 2 D), and 45 days of 5-day IF ( Figure 2 E) all did. 50 days of 5-day IF slightly reduced lifespan extension compared to 45 days ( Figure 2 F). Additionally, 50 days of the IF diet led to some early death before the IF switch to ad libitum conditions. Therefore, there appears to be a duration-specific and age-specific window where IF can exert its beneficial effects.

For display purposes, in (A), (B), and (F), the ad libitum condition is the same data in each separate graph (solid black line). In (C), (D), and (E), the ad libitum condition is the same data in each separate graph. In (G), the ad libitum condition is the same data in each separate graph. See also Figure S3

(G) Switching 5-day IF flies to ad libitum after 30 days of the regime resulted in significant lifespan extension compared to ad libitum controls (p = 0.019; log rank test). Switching flies from ad libitum food for the first 20 days of life to 5-day IF spanning days 20–45 (25 days) and then back to ad libitum food resulted in significant lifespan extension compared to ad libitum controls (p = 0.045; log rank test). n > 145 flies per condition. Grey dashed line indicates the switch point, after which the lifespan curves were reset and deaths before this point were censored.

(F) Switching 5-day IF flies to ad libitum after 50 days of the regime resulted in significant lifespan extension compared to ad libitum controls (p = 0.00046; log rank test). n > 145 flies per condition.

(E) Switching 5-day IF flies to ad libitum after 45 days of the regime resulted in significant lifespan extension compared to ad libitum controls (p = 1.4 × 10 −14 ; log rank test). n > 150 flies per condition.

(D) Switching 5-day IF flies to ad libitum after 40 days of the regime resulted in significant lifespan extension compared to ad libitum controls (p = 1.2 × 10 −13 ; log rank test). n > 165 flies per condition.

(C) Switching 5-day IF flies to ad libitum after 30 days of the regime resulted in significant lifespan extension compared to ad libitum controls (p = 3.6 × 10 −10 ; log rank test). n > 190 flies per condition.

(B) Switching 5-day IF flies to ad libitum after 20 days of the regime did not affect lifespan compared to ad libitum controls (p = 0.057; log rank test). n > 195 flies per condition.

(A) Switching 5-day IF flies to ad libitum after 10 days of the regime did not affect lifespan compared to ad libitum controls (p = 0.15; log rank test). n > 195 flies per condition.

Because the 2:5 diet reduced egg production, we experimentally tested the role of reduced fecundity in the increased longevity from IF. We performed IF on virgin females, which produce fewer eggs than mated females [], and on sterile females carrying the dominant ovomutation, which blocks oogenesis []. Lifespan was significantly extended in virgin wfemales after IF ( Figure 1 E). Additionally, whereas ovofemales initially exhibited a greater sensitivity to 5-day IF compared to outbred wflies (see Figures 1 A and 1B), 5-day IF switch ovoflies lived significantly longer than controls ( Figure 1 F). To rule out a selection bias for the most starvation-resistant ovoflies, we also performed shorter duration IF on these flies. Whereas 3-day IF switch in ovoflies had no significant effect on lifespan ( Figure S3 E), 4-day IF switch significantly extended lifespan ( Figure S3 F) but without any excess early mortality. Therefore, similar to DR [], the pro-longevity phenotype observed after short-term early-life IF is not mediated by reduced fecundity.

IF induced a greater lifespan extension in female flies compared to males. We hypothesized that this may have been due to reduced fecundity, a hallmark of pro-longevity interventions, such as DR []. We therefore performed egg counts during the fasting/fed period. Fecundity was indeed all but abolished during the fasting period and was restored after re-feeding ( Figure S1 C).

Male flies also benefited from the IF switch regime. Male flies are more starvation sensitive than females []. Therefore, males were exposed to 4-day IF for 25 days. This resulted in significant lifespan extension ( Figure 1 D). We also tested 2-day IF and 3-day IF on males, but there was no effect ( Figures S3 C and S3D). Therefore, both sexes can benefit from early-life dietary intervention with increased longevity, and these effects are dependent on the duration of the fasting period.

To rule out strain specificity of our results, we performed IF on the isogenic lab strain w. Lifespan was significantly extended in wafter 30 days of 5-day IF ( Figure 1 C). wfemales initially exhibited a greater sensitivity to 5-day IF compared to outbred wflies ( Figures 1 A and 1B), and so, to rule out a selection bias for the most starvation-resistant flies, we also applied shorter duration IF to these flies. Whereas 3-day IF switch had no significant effect ( Figure S3 A), 4-day IF switch significantly extended lifespan in wflies ( Figure S3 B). Importantly, the early-life mortality disappeared in these flies, whereas the longevity phenotype remained.

Less severe IF regimes might also result in positive outcomes compared to 5-day continuous IF. We therefore next examined the effects of shorter fasting duration periods with 2-day, 3-day, and 4-day continuous IF throughout adult life. None of these regimes had a significant effect on lifespan ( Figures S2 A–S2C). When the same regimes with an additional IF switch condition were examined, 2-day and 4-day IF switch had no significant effect, whereas the 3-day IF switch condition lived significantly longer ( Figures S2 D–S2F). Increasing the caloric content of the food is another way in which the potential severity of IF may be offset. When flies were fed food with twice the amount of yeast (“2SY”) [], fasting for 2 days per week throughout adult life significantly increased lifespan ( Figure S2 G).

To test directly for an age-specific benefit of IF, we repeated the 5-day continuous IF experiment and included an additional IF group that was returned to ad libitum feeding after 30 days of the IF regime (labeled “IF switch”). Lifespan was again shortened by 5-day continuous IF throughout life ( Figure 1 B), but the IF switch group had significantly extended lifespan compared to the ad libitum controls. An independent repeat of this experiment confirmed these results ( Figure S1 B). Thus, continuous exposure to the 2:5 diet induced mortality in older flies, but the same dietary regime for the first 30 days of adulthood resulted in significant lifespan extension in female flies, indicating a long-lasting “memory” effect of this early-life intervention.

We hypothesized that the periods of starvation in previously published IF regimes may have been insufficient in duration to influence survival []. We therefore began by fasting female flies by exposing them to nutrient-free agar gel for 5 consecutive days per week (5-day IF—the 2:5 diet) throughout life and found that this led to earlier onset of age-related mortality and significantly shortened lifespan compared to ad-libitum-fed controls ( Figure 1 A). Mortality rates were lower during 2-day feeding periods in the IF flies, an effect that persisted into the first 3 days after food was withdrawn. Because 5 days of continuous starvation was detrimental, we next investigated the effects of 5 days non-continuous starvation per 7 days by fasting for 3 days, feeding for 1 day, fasting for 2 days, and feeding for 1 day and found that this regime also significantly shortened lifespan, although with a mildly protective effect up to day 50 ( Figure S1 A). Taken together with the observation that 5-day continuous IF did not start to induce mortality until the flies were ∼30 days old ( Figure 1 A), these findings suggested that the effects of IF might be age specific. Fly feeding rate declines with age [], and older flies may therefore be more sensitive to starvation and exhaustion of energy stores. Any beneficial effect of IF may therefore be lost or even reversed later in life.

(G) Every-other-day fasting for the first 30 days of life (EOD switch) did not affect lifespan compared to ad libitum controls (p = 0.096; log rank test), whereas IF switch significantly increased lifespan compared to ad libitum controls (p = 0.017; log rank test). n > 166 flies per condition. Grey dashed line indicates the “switch” point at 30 days (25 days for males), after which the lifespan curves were “reset” and deaths before this point were censored.

(F) Sterile ovo D flies exhibited a greater sensitivity to 5-day IF compared to outbred w Dah flies (see A and B). Switching 5-day IF ovo D flies to ad libitum after 30 days of the regime resulted in significant lifespan extension compared to ad libitum controls (p = 0.0048; log rank test). n > 160 flies per condition.

(E) Switching 5-day IF virgin female flies to ad libitum after 30 days of the regime resulted in significant lifespan extension compared to ad libitum controls (p = 0.012; log rank test). n > 170 flies per condition.

(C) Isogenic w 1118 flies exhibited a greater sensitivity to 5-day IF compared to outbred w Dah flies (see A and B). Switching 5-day IF w 1118 flies to ad libitum after 30 days of the regime resulted in significant lifespan extension compared to ad libitum controls (p = 3.0 × 10 −10 ; log rank test). n > 180 flies per condition.

(B) Switching 5-day-fasted flies to ad libitum after 30 days of the IF regimen resulted in significantly increased lifespan compared to ad libitum controls (p = 0.0022; log rank test; n > 145 flies per condition).

Discussion

6 Wei M.

Brandhorst S.

Shelehchi M.

Mirzaei H.

Cheng C.W.

Budniak J.

Groshen S.

Mack W.J.

Guen E.

Di Biase S.

et al. Fasting-mimicking diet and markers/risk factors for aging, diabetes, cancer, and cardiovascular disease. IF can be highly effective against age-related diseases, including diabetes, cancer, and cardiovascular disease []. Although IF research is still in its infancy, the mechanisms mediating the health improvements seen during and after IF are starting to be discovered.

26 Grandison R.C.

Wong R.

Bass T.M.

Partridge L.

Piper M.D.W. Effect of a standardised dietary restriction protocol on multiple laboratory strains of Drosophila melanogaster. 33 Le Bourg E.

Médioni J. Food restriction and longevity in Drosophila melanogaster. In this study, we clarified the effects of IF on health and lifespan in Drosophila. In line with previous studies [], we found that IF for short fasting periods over the duration of the adult female lifespan had no beneficial effect (e.g., 2 fasted days per week). IF shortened lifespan when the fasting period was lengthened, e.g., 5 fasted days per week (the 2:5 diet). However, when flies were switched to ad libitum conditions after 30 days of a prolonged IF regime, median lifespan could be extended by as much as 10%. The pro-longevity effect of short-term, age-specific IF was not sex specific but was more effective in females, was consistent over a range of genotypes, was not due to a DR-like reduction in post-IF food intake, and was not dependent on reduced fecundity, indicating mechanisms other than reproductive trade-offs.

56 Gems D.

Partridge L. Stress-response hormesis and aging: “that which does not kill us makes us stronger”. 57 Arking R. Strategies for stage-specific extension of longevity. Remarkably, IF during early adulthood, and also during mid-life, was sufficient to extend lifespan, indicating a “memory” effect. Increased resistance to multiple stresses after IF was also an indication of significantly improved overall health. Hormesis is a phenomenon by which “low-level” toxic stress elicits response mechanisms that protect against similar but higher level stresses associated with aging []. Given that intermittent starvation in early life led to increased post-IF starvation resistance in addition to increased lifespan, hormesis could play a role. Although we cannot rule out the possibility of a hormetic effect of IF, the pro-longevity effect of IF restricted to early and mid-life, and the opposing lifespan-shortening effect of IF restricted to late life, suggests an age-specific effect of IF and that other mechanisms may be more relevant. Further work is necessary to elucidate the potential hormetic effects of IF, as well as assessment of longevity interventions during specific fly life stages, particularly early versus late interventions, as this remains a relatively underexplored area of aging research [].

The increased TAG levels, stress resistance, and longevity are also potentially a direct consequence of post-IF hyperphagia. Intriguingly, 42-day-old IF switch flies were more starvation resistant compared to ad libitum controls but had lower TAG levels, indicating a mechanism other than increased fat storage. A more detailed study of feeding behavior during IF and post-IF would be necessary to investigate this hypothesis.

29 Regan J.C.

Khericha M.

Dobson A.J.

Bolukbasi E.

Rattanavirotkul N.

Partridge L. Sex difference in pathology of the ageing gut mediates the greater response of female lifespan to dietary restriction. IF influenced the female gut, with a distinct improvement in measures of age-related pathology, including a preservation of the “youthful” phenotype, i.e., reduced ISC activity; fewer layers of nuclei, indicating reduced tumor formation; and improved gut barrier function []. However, gut length and gut width were no different from those of controls, suggesting a recovery of gut functionality without confounding effects of shorter/narrower guts, which may result in indirect (functional) DR. Indeed, the increased lipid content in 60-day-old flies despite early-life fasting points to a protective effect from the age-related decline in gut function. However, further analysis probing the absorptive capacity of the gut during aging is necessary before improved gut health and absorption can be linked. The increased TAG levels seen in 5-day IF switch flies, but not in 3- or 4-day IF switch flies, may also explain the differing effectiveness of each of these regimes.

58 Shin S.C.

Kim S.H.

You H.

Kim B.

Kim A.C.

Lee K.A.

Yoon J.H.

Ryu J.H.

Lee W.J. Drosophila microbiome modulates host developmental and metabolic homeostasis via insulin signaling. Increased TAG is also observed with loss of fly-associated bacteria [], which is consistent with the changes in TAG seen with IF switch flies. We therefore measured relative bacterial abundance in post-IF females and found that, despite higher post-IF food intake, relative bacterial abundance was reduced in IF switch flies. Longer periods of fasting appear to reduce total bacterial load, and this is associated with improved gut health and lifespan extension. However, further work is required to elucidate whether the beneficial effects of IF are due to reduced microbial load and should assess whether IF would lead to increased longevity in germ-free conditions.

53 Bjedov I.

Toivonen J.M.

Kerr F.

Slack C.

Jacobson J.

Foley A.

Partridge L. Mechanisms of life span extension by rapamycin in the fruit fly Drosophila melanogaster. 53 Bjedov I.

Toivonen J.M.

Kerr F.

Slack C.

Jacobson J.

Foley A.

Partridge L. Mechanisms of life span extension by rapamycin in the fruit fly Drosophila melanogaster. 31 Wei M.

Fabrizio P.

Hu J.

Ge H.

Cheng C.

Li L.

Longo V.D. Life span extension by calorie restriction depends on Rim15 and transcription factors downstream of Ras/PKA, Tor, and Sch9. 32 Honjoh S.

Yamamoto T.

Uno M.

Nishida E. Signalling through RHEB-1 mediates intermittent fasting-induced longevity in C. elegans. Finally, we probed the mechanisms underlying the pro-longevity properties of IF. We performed pharmacological and genetic epistatic analysis to demonstrate that IF acts independently of the TOR pathway, because rapamycin and the IF regimen acted additively to extend lifespan. Similarly, Bjedov and colleagues [] found that the pro-longevity effects of rapamycin and DR were additive. Global expression of a constitutively active S6K did not attenuate the IF-induced lifespan extension, whereas the lifespan-extending effects of rapamycin were abolished by this method []. Our results agree with findings in yeast and nematodes that IF-induced lifespan extension can act independently of TOR signaling []. It should be noted that we would not recommend the 2:5 diet, cycles of 5 consecutive days of water-only fasting per week, in humans.

In summary, we have generated and optimized a reliable and robust regime of IF for lifespan extension in flies. We conclude that IF during early life can robustly increase lifespan in a TOR-independent manner and likely acts by preserving gut health. Importantly, the beneficial effects of short-term IF were long-lasting, indicating that even brief IF periods may have long-lasting health benefits, a phenomenon that should be subject to further investigation in mammals.