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To evaluate the effect of combination antiretroviral therapy (cART) on semen quality.A longitudinal cohort study.The HIV outpatient clinic of the Academic Medical Centre in Amsterdam, the Netherlands.A cohort of 34 male patients with different estimated duration of HIV-1 infection, who were about to start various combinations of cART.Blood and semen analyses before the start of cART and 4, 12, 24, 36 and 48 weeks thereafter.We examined the effect of cART on semen parameters by a repeated measurements procedure using a mixed-effects model.The median period of follow up was 48 weeks (interquartile range 33–52 weeks). Five patients used thymidine analogue-containing cART, 23 used tenofovir-based cART, six used other regimens. At all timepoints the percentage of progressively motile spermatozoa was low according to WHO criteria, and it decreased significantly from 28 to 17% during follow-up (P = 0.02). All other semen parameters were in the normal range and remained stable.cART negatively affected the percentage of progressively motile spermatozoa. Whether this reduced motility affects the chances of fathering a child or leads to an increased need for artificial reproductive techniques is at present unknown.