Purpose <p>Fundus parameters can be used to quantify masculinity or femininity as a fundus sex index (FSI) ranging from 0 to 1. The purpose of this study was to investigate the association between smoking, hypertension, diabetes, and FSI in the Kumejima population study</p> Study design <p>Prospective cross-sectional observational population study</p> Methods <p>Using color fundus photographs obtained from the Kumejima population study, 1653 healthy right eyes with reliable fundus parameter measurements were included. The tessellation fundus index R/(R + G + B), red-green-blue intensity in eight locations around the optic disc and foveal region, optic disc ovality ratio, papillomacular angle, and retinal vessel angles were quantified using ImageJ. The FSI was calculated using machine learning from the 42 fundus parameters, and the Mann–Whitney U-test was used to assess whether there were differences in the FSI depending on the presence or absence of smoking, hypertension, and diabetes</p> Results <p>The mean age of the 838 men and 815 women included in this study was 52.8 and 54.0 years, respectively. The FSI of the smoking group was lower than of the non-smoking group overall (P &lt; 0.001). The FSI of the hypertension and diabetes groups was significantly higher than of the non-hypertension (P = 0.005) and non-diabetes (P = 0.017) groups only in women</p> Conclusion <p>In the Kumejima population study of healthy eyes in individuals aged 40 years or older, the fundus tended to be more feminine in women with hypertension or diabetes than in those without these conditions</p>