Nearly twice as many F/G-fed females died in OPAs compared with controls and a complex effect of the F/G diet on reproduction was also observed in OPAs. Specifically, F/G females exhibited higher reproductive success early in the study, but a decreasing rate of reproduction as the study progressed. Previous studies demonstrate a positive association between adiposity and reproductive success in females, which is thought to be due to the availability of energy reserves18. F/G feeding during the pre-OPA period may have increased adiposity (while not increasing body weight), which could underlie the early elevation in reproductive success. Subsequent reproductive decline and rise in mortality observed in the latter part of the OPA testing could be explained by the mounting physiological insults of the F/G diet interacting with the extreme energetic pressures of simultaneous gestation and lactation, which elevate metabolism by 18–25%19. Future studies designed to track these parameters may help in elucidating the relationship between the F/G diet, adiposity, reproductive success and mortality.

Overall, F/G-fed males were outcompeted by control animals as measured by competitive ability and reproduction. Though death rates of males were higher than females due to intra-sexual competition, they did not differ between dietary groups. Therefore, it is likely that the lower reproduction of F/G males was due to their decreased ability to defend territories, though other mechanisms may also have decreased reproductive output. Previous work in our system has indicated territorial males sire ≈80% of all pups within an enclosure, as F/G feeding decreased both territorial acquisition and reproduction by ≈25%, it is reasonable to assume that the majority of the reproductive decline is driven by decreased competitive ability, but that a portion of the difference could be due to other reproductive impairments15. Previous work demonstrates increased male adiposity due to added sugar at higher exposure levels (for example, Jurgens et al.8); therefore, a plausible explanation is that F/G-fed males could have increased adiposity, despite not having different body weights, leading to a disadvantage in the physical contests required to acquire territories and mates.

The sex-specific nature of our OPA findings is interesting though not unprecedented. OPAs revealed a similar mortality pattern, when females harbouring the selfish genetic element known as the t complex experienced an increased risk of mortality while males did not15. Likewise, at both cousin and sibling-levels of inbreeding reproductive output in males was impacted to a much higher degree than that of females13,14. These findings are inline with the notion of a trade-off between reproduction and longevity (for review see Partridge et al.20). Given that the energetic allocation toward offspring is much higher for females than for males it is not surprising that females might incur increased mortality due to a nutritional manipulation, while males would be more likely to decrease reproductive output.

The only pre-OPA fasting plasma measure that was associated with the decreased reproduction and increased mortality exhibited by F/G-fed animals was total cholesterol, as no difference was seen in plasma glucose, insulin or triglyceride concentrations. These data suggest that the F/G-fed animals may be suffering from dysregulated lipid metabolism before OPA entrance and supports our hypothesis that differential adiposity may have existed between dietary treatments. In the future, tracking and fractionating cholesterol of animals within OPAs and determining if increased plasma values are predictive of OPA outcomes would be highly informative.

Impaired glucose clearance rates of F/G-fed females compared with controls before OPA entrance may reflect a yet to be identified physiological alteration, possibly loss of endocrine capacity, that underlies and predicts increased risk of death within OPA enclosures. However, this impairment in glucose clearance and the underlying physiological alterations that it biomarks is masked within 2 weeks of residing in OPAs, well before the majority of deaths have occurred. Nevertheless, disrupted glucose metabolism throughout the developmental period from weaning into adulthood could cause lasting physiological problems and the glucose disruption may persist in individuals within OPAs, but at levels below the detection limits of current assays. No diet induced differences were seen in regards to male glucose clearance (though both F/G and control-fed males exhibited slower clearance relative to control females) and differential mortality between groups did not occur indicating that perhaps differential glucose clearance is the basis of this sex-specific finding. That both dietary groups, as well as both sexes, markedly increased their rates of glucose clearance after entering OPAs is likely due to increased physical activity permitted by the larger living area as well as the increased energetic demands of reproductive and social activities21. As elevated activity leads to increased glucose disposal, metabolic impairments may be masked, making it difficult to establish connections between the pre-OPA alterations in metabolism to the adverse impacts observed in the OPA.

The increased rates of mortality and decreased reproduction observed in this study represent the lowest level of added sugar consumption shown to adversely affect mammalian health. These adverse organismal-level findings are detectable while standard clinical measures are either unaltered (body weight, glucose, insulin and triglycerides), or exhibit complicated patterns (cholesterol and glucose tolerance), indicating that the available clinical measures used are not of sufficient sensitivity to reflect the physiological impairments leading to early death in females and reproductive declines in males.

With complex organismal-level endpoints like survival, reproduction and competitive ability it is highly unlikely that a single molecular mechanism acting in isolation is responsible for producing the observed phenotype. It is more likely that a variety of mechanisms at the molecular, cellular and higher levels are acting in concert to alter phenotypes. Our glucose clearance and cholesterol results likely represent part of the mechanistic cascade that leads to the decreased survival, reproduction and competitive ability caused by F/G feeding; however, they do not represent its entirety. Clearly more mechanistic work is needed to elucidate the underpinnings of our findings, but current searches into the causes of added sugar-induced diseases are solely focused on extreme exposure levels and therefore may not aid in characterizing effects at human-relevant exposures. The characterization of mechanisms at relevant doses will be more difficult, as high doses are typically used to ‘push the system’ and decrease the amount of time and number of animals required, but the information garnered will be more readily translated to the human condition. Regardless of the mechanisms involved our study characterizes the ultimate impacts of F/G-feeding mice at human-relevant levels.

We detected substantial adverse outcomes due to an added sugar exposure consisting of a 1:1 ratio of fructose and glucose amounting to 25% kcal. Our results provide evidence that added sugar consumed at concentrations currently considered safe exerts dramatic adverse impacts on mammalian health22,23. Many researchers have already made calls for reevaluation of these safe levels of consumption24. Although others have advocated for more drastic regulatory measures to curtail sugar consumption25. Our study provides experimental data directly supporting mammalian adversity at these ‘safe levels’ and provides a model system in which to investigate the molecular and physiological mechanisms causing this organismal adversity.

Though OPAs detected large differences in reproductive output and survival between F/G-fed and control animals, the results from our studies are likely conservative. First, OPAs were terminated at 34 weeks because of the common, diet independent, high rates of male attrition; it is likely that if the study continued for the entirety of the mouse lifespan that reproductive outputs between the treatments would have continued to diverge. Second, at the start of OPA assessment all animals were provided the same sugar enriched diet, because as yet we have no way to keep animals on their respective diets during OPAs. This means that all of the adverse effects of the F/G diet are a consequence of exposure before OPA entrance. Third, our F/G diet was based on a modified chow and not a refined diet, meaning that our F/G-fed animals showed impairment despite having the remainder of their diet being highly nutritive, whole grain-based, with optimum mineral and vitamin composition, analogous to human subjects consuming a healthy diet plus the equivalent of ≈3 cans (354 ml each) of soda per day.

By directly assessing the impacts of a treatment on the performance of house mice in OPAs, we are capable of bridging the environmental relevance and longitudinal nature of human studies with the controllability and experimental power of animal models. The data within this paper along with other similar successes using OPAs suggest that this and similar approaches will be an important tool in the detection and quantification of adversity caused by a wide array of treatments13,14,15. Once adversity is detected in OPAs and an adverse phenotype characterized, reductionist techniques can be applied to discover the mechanism and thus provide human translatability. As output measures are similar across experiments, OPAs allow for the direct comparison of disparate treatments. For example, our data indicate that the F/G diet is as detrimental to male reproduction as cousin-level inbreeding (Supplementary Fig. S2).

Currently, there is a great need for sensitive toxicity assessment methods that work across a broad range of experimental manipulations. This need is particularly strong for both pharmaceutical science where 73% of drugs that pass preclinical trials fail due to safety concerns and for toxicology, where shockingly few compounds receive critical or long-term toxicity testing26,27. Likewise, with health-care costs spiralling out of control, partially due to an epidemic of diet-related non-communicable diseases, one could use this approach to identify other dietary components that may link to adverse outcomes (or benefits). Assuredly, the revelation that added sugar consumption at human-relevant concentrations increases death rates and decreases both competitive ability and reproductive output is evidence that OPAs have the potential to identify disease risks that escape notice by standard clinical measures when tested at relevant-human exposures.