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The aim of this study was to assess the acute effect of preoperative RT for rectal cancer on endocrine testicular function.Preoperative radiotherapy (RT) enhances local control and cancer-specific survival in patients treated for rectal cancer. In case series, a negative acute effect on Leydig cell function has been reported.This prospective cohort study included 168 males with rectal or prostate cancer stage I-III. Males treated with preoperative RT and surgery for rectal cancer formed the exposed group (n = 93). Males treated with surgery alone were assigned to the unexposed group (n = 75). The androgen levels were assessed at baseline and after preoperative RT. The exposure was quantified with the treatment planning system to estimate the cumulative testicular dose (TD). The risk of low T (serum T < below 8 nmol/L) was the primary endpoint. Secondary endpoints were serum testosterone (T), bioavailable T, luteinizing hormone (LH), and the LH-T ratio.The baseline levels of androgens were not related to exposure status or type of cancer. The proportion of low T increased from 14.6% at baseline to 35.4% after RT, relative risk 2.41 (95% CI 1.57 to 3.71, P < 0.001). Preoperative RT resulted in a significant decrease of serum and bioavailable T and a significant increase of LH and LH-T ratio. The decline in serum and bioavailable T was related to the TD.Preoperative RT for rectal cancer results in dose-dependent primary testicular failure increasing the risk of hypogonadism at the time of surgery by 2.4 times (number needed to harm = 5).