Abstract Study question What impact do smoking and vaping have on ovarian reserve markers Anti-Mullerian Hormone (AMH) and Follicle-Stimulating Hormone (FSH)? Summary answer Smoking and vaping negatively affect AMH and FSH levels in women, with significant age-related differences in ovarian reserve markers observed. What is known already Previous studies have established a link between smoking and accelerated ovarian follicular depletion, evidenced by lower serum AMH levels and reduced antral follicle count (AFC). Smoking is known to increase FSH levels indirectly by affecting pituitary function. However, the impact of vaping, a growing trend especially among younger individuals, on ovarian reserve markers like AMH and FSH remains under-researched and requires comprehensive investigation to understand its full implications on reproductive health. Study design, size, duration This cross-sectional study analysed medical history and serum AMH and FSH concentrations from capillary blood samples of 8,340 women aged 21-45. Participants were stratified by 5-year age intervals and excluded if they had reproductive health conditions or used hormonal contraception. Samples were analysed from individuals using an at-home hormone testing service, with statistical analysis including unpaired t-tests and Cohen’s d for effect size, focusing on smokers/vapers versus non-smokers/non-vapers. Participants/materials, setting, methods The study included UK women aged 21-45 providing capillary blood samples for serum AMH and FSH measurement using an at-home testing service. Exclusions applied to those with reproductive conditions or on hormonal contraception. Smoking and vaping status were self-reported through an online health assessment. AMH and FSH concentration differences were analysed via unpaired t-test; a p-value of < 0.05 was considered statistically significant and mean values±SD were reported. Main results and the role of chance Among 8,340 participants, 312 smoked and 653 vaped. Lower serum AMH values were observed in smokers and vapers in all age groups, with significant differences at ages 31-35 for smokers (20.73±16.23 vs 25.37±19.80 pmol/L in smokers and non-smokers respectively; p = 0.003) and 36-40 for vapers (13.65±13.34 vs 16.88±14.32 pmol/L in vapers and non-vapers respectively; p = 0.01). No significant differences in serum FSH concentration were found among vapers, however, FSH levels were higher in smokers across all ages. This was significant in individuals aged 21-25 (8.01±2.52 vs 6.87±3.33 IU/L in smokers and non-smokers respectively; p = 0.02) and 31-35 (8.33±3.31 vs 7.70±3.51 IU/L smokers and in non-smokers respectively; p = 0.04). These results suggest a detrimental effect of smoking and vaping on ovarian reserve markers, with statistical significance indicating the role of chance is minimal in the observed differences. Limitations, reasons for caution Self-reported smoking/vaping status may introduce bias. The study does not report the duration or frequency of participants’ smoking and vaping habits. The study’s cross-sectional design limits causality inference, however it provides a valuable insight for future longitudinal research to explore these relationships over time and explore underlying mechanisms. Wider implications of the findings These findings highlight the potential risks of smoking and vaping on women’s reproductive health, emphasising the need for awareness and preventive measures, especially among younger populations and the increased use of vaping. Trial registration number not applicable