Hypogonadal obese men find it difficult to lose weight. We investigated whether the modification of macronutrient intake can alter testosterone levels independently of the body mass index. Fasted overweight or obese fertile men were asked to consume meals of polyunsaturated fats (PUFA), monounsaturated fats (MUFA), refined carbohydrates (CHO, orange juice, OJ), whey and egg albumin and mixed meals of PUFA and CHO, PUFA and egg albumin, and CHO and egg albumin. Blood was collected at fasting, then hourly for 5 h and analysed to determine the levels of testosterone and other hormones. We found PUFA and MUFA or a mixed meal of PUFA and CHO significantly reduced serum testosterone production to a similar degree over a 5 h period. PUFA decreased serum testosterone levels by 3.2 nmol/L after 1 h compared to baseline (p = 0.023), with this suppression remaining significant up to 5 h postprandially (2.1 nmol/L / p = 0.012). The net overall testosterone levels were reduced by approximately 10 nmol/L × / h by PUFA, MUFA and PUFA combined with CHO. CHO alone had little effect on testosterone levels, whereas egg albumin was able to increase them (7.4 cf 2.0 nmol/L × / h). Therefore, for men wishing to optimize their testosterone levels, it may be wise to avoid a high fat intake, drink liquids such as water or OJ or even consider fasting. ANZCTR, Australia / ACTRN12617001034325.