Objective: Transvaginal ultrasound-guided aspiration (TUA) is a procedure which can be used for the reduction of twins post-fixation in the mare. The aim of this study was to evaluate the effect of the age of mares and the day of gestation on the outcome of TUA treatment. Material and methods: In 88 mares, diagnosed pregnant with twins, TUA of the yolk sac or allantoic fluid was performed between day 30 and 62 of gestation. Mares were aged 3–22 years. Ultrasonographic examination for a viable singleton pregnancy was performed by referring veterinarians 5–7 days and 4 weeks after TUA. Based on reported findings, effects of age and day of gestation on pregnancy rates were evaluated. Results: Four weeks after TUA, 67 % of the cases resulted in a viable singleton pregnancy. Five to 7 days after TUA treatment, the success rate was 74 %. The gestational period did not affect the outcome, irrespective of the age of the mare. In contrast, success rates decreased with increasing age of the mares (84 %  7 years vs. 67 % 8–14 years vs. 57 %  15 years). In mares aged 8−14 years, a decrease in singleton pregnancies was observed, if TUA was performed after day 35 of gestation. Success rates were slightly higher, if twin vesicles were localized within separate uterine horns (73 %) as compared to the same horn (66 %). Differences in singleton pregnancy rates were not statistically significant (p > 0.05). Conclusions and clinical relevance: TUA was found to be an effective procedure for reduction of twin pregnancies performed at days 30–62 of gestation. Success rates for singleton pregnancies were high for young mares  7 years old (84 %) and middle aged mares treated before day 36 of pregnancy (74 %). Duration of pregnancy at the time of TUA did not have a major impact on the outcome. Nevertheless, the procedure should optimally be performed around days 32–35 of pregnancy to allow for the possibility of natural reduction before treatment and rebreeding in case of a total pregnancy loss after TUA.