Abstract Study question Does paternal pre-conception consumption of caloric or non-caloric sweeteners (NCS) coupled with high fat diets (HFD) predispose offspring to early puberty onset in mice? Summary answer Paternal prenatal consumption of the NCS Rebaudioside A(RebA) resulted in early puberty onset in female offspring only compared to water and caloric sweetener groups. What is known already A growing body of evidence suggests that the paternal diet also influences health and disease onset in offspring. Early puberty onset is associated with metabolic syndrome, type 2 diabetes, and cardiovascular disease later in adulthood. NCS are often promoted as heathier alternatives to caloric sweeteners. Evidence examining the impact of NCS on male reproductive function and offspring health outcomes, including pubertal timing are lacking. Few studies have shown maternal HFD can contribute to early puberty onset in female offspring however to our knowledge this is the first study to investigate paternal diet and offspring reproductive outcomes. Study design, size, duration 32 6-week-old C57/BL6 male mice were randomly assigned to receive a control diet (CD) or HF plus water (Con), fructose (Fr), Acesulfame K (Ak) or RebA). After 8 weeks, mice were mated. Litters were standardised to 2 males and 2 females per group and fed a standard laboratory diet. Participants/materials, setting, methods Puberty onset was measured in females by vaginal opening (from postnatal day (PN28) and in males by preputial separation (from PN35). Oral glucose tolerance tests were carried out at PN70. Mice were culled from PN80. Data were analysed by repeated measures or 2-way ANOVA. Main results and the role of chance There were no significant differences in birth weight, sex ratio or organ weights across groups. There was an overall effect of paternal NCS on male but not female offspring weightCR1 at cull(p < 0.05), with male CDRebA offspring having increased BW (29.52g±1.5) compared with CDAk at PN80 (26.4g±3; p < 0.05). Females from paternal HF and CD RebA group CR2 entered puberty significantly earlier (31.8d±1) than Fr (34d±1;p<0.01) and Con (34±1;p<0.05) groups. Male offspring from HFAk had lower glucose AUC (913±31) compared with CDCon (1289±48;p<0.001). Female offspring from CDRebA (1263±84) and CDFr (1273±67) groups had lower glucose AUC compared with CDCon group (1445±56;p<0.05). Limitations, reasons for caution This study was carried out on mice therefore direct translatability to humans is limited. examining the impact of paternal NCS intakes on molecular pathways in the ovary and testes to assess offspring reproductive health later in life and to evaluate pregnancy success following mating would essential for providing further insight. Wider implications of the findings The present study adds to the experimental evidence to date suggesting that NCS may not be beneficial alternatives to sugar-sweetened products consumed preconceptionally on the health of the offspring. This study also highlights the importance of including paternal diet in research when looking at transgenerational effects. Trial registration number not applicable