Over recent years a consistent increase in male infertility has been reported. Research into the underlying aetiologies have suggested a reduction in semen quality, possibly related to lifestyle, occupational or environmental exposures, and that sperm DNA strand breaks and base damage might be biomarkers of male infertility. Therefore, the aims of this study were to examine the variations of semen quality parameters and DNA damage in human sperm and associations between these factors and lifestyle factors and environmental exposures. Twenty participants were initially enrolled in the study and 15 of them completed the entire study. Two hundred and six wide ranging food, lifestyle and occupational questionnaires, 206 semen samples and 59 blood samples were collected from the participants on 12 separate visits during the 6-month study period. The sperm parameters were assessed manually and by computer assisted sperm analysis, Sperm DNA double strand break damaged was visualised by a neutral Comet assay and scored manually to generate a genetic damage indicator (GDI). The levels of the DNA lesion, O6-alkylguanine (O6-alkylG) in sperm and buffy coat DNA samples were quantified using a novel assay. The study showed that total sperm count and sperm concentration were the most variable semen parameters, while the lowest variable parameter was % vital sperm. The Comet assay was shown to have very good reproducibility and repeatability. At baseline, the GDI values of the 20 participants ranged from 29 to 166, and these levels were remarkably consistent over the longitudinal 6 month study period (29+/-3 and 163+/-6 respectively). There was a significant negative correlation between % vital sperm and GDI (R=minus0.57 P=0.01). The novel O6-alkylG quantitation assay was optimised and showed a range of levels in sperm DNA. These did not correlate with the levels in the corresponding buffy coat DNA nor with the corresponding GDI, the basis of which may be related to the activity of various DNA repair pathways. However, the assay was found to be simple and reproducible and could be used to assess the overall levels of O6-alkylG in DNA of human sperm or any other tissues in future studies. Some statistically significant positive or negative correlations were found between a few of the many diet, lifestyle and occupational parameters studied, but the numbers were in all cases too small to have any real confidence. This study is therefore to be seen as a prelude to a much larger study requiring substantially more participants.