For artificial insemination (AI) in the pig, semen is routinely maintained at room temperature for 2–4h prior to extending—to reduce the cooling damage to sperm during cryopreservation. In the sheep industry, however, semen is diluted and cooled immediately after collection. This trial evaluated the effect of a 4h pre-incubation period for semen at room temperature on the subsequent quality parameters of ram sperm prepared for AI. Immediately following collection, ram semen was divided in 2 aliquots—one was left undiluted for 4h at room temperature (20°C; pre-incubation) and the other (control) was diluted with an egg-yolk-based extender and either cooled to 5°C (n=8 different ejaculates) for short-term fresh conservation or cryopreserved (n=6 different ejaculates). After 4h at room temperature, the pre-incubated semen was then diluted and either cooled to 5°C or cryopreserved, as was the control. Sperm motility, viability and chlortetracycline (CTC) pattern distribution of the pre-incubated semen were compared to the control. For fresh semen conserved at 5°C, total sperm motility and the proportion of CTC pattern F sperm (referring to non-capacitated, non-acrosome reacted cells) were reduced by the 4h incubation at room temperature, compared to the control. The effect of pre-incubation at room temperature was more evident in the cryopreserved semen in terms of total and progressive sperm motility, with the viability being reduced following pre-incubation. For the cryopreserved semen, the percentage of CTC pattern F sperm declined, while the pattern of AR sperm (referring to acrosome-reacted cells) increased, compared to the controls. In conclusion, pre-incubation of ram semen for 4h at room temperature prior to preparation for AI is not beneficial to the subsequent functionality of the sperm. Furthermore, this pre-incubation period is more harmful to frozen-thawed than to fresh-cooled sperm.