The aim of this study was to evaluate the impact of successive bovine testicular punctures using different needle sizes. Fifteen bulls were submitted to testicular needle aspiration (TNA) in the left and right testis using 18-gauge (40×12mm) or 22-gauge (25×7mm) needles, respectively, once every 30 days. Animals were randomly divided into three groups, which were submitted to bilateral orchiectomy two days after the last puncture. Group 1 (G1): only one puncture (n=5); Group 2 (G2): three consecutive punctures in a period of three months (n=5); Group 3 (G3): six consecutive punctures in a period of 6 months (n=5). Fragments from the medial portion of the testicular parenchyma were excised and fixed in Bouin's fluid for histological analysis. No differences were observed in the percentage of seminiferous tubules degeneration between G1, G2 and G3 (P>0.05). Higher amounts of erythrocyte were found in G1 and G2 groups compared to G3, in the intra-and intertubular tissue (P<0.05). There was no interaction between the needle gauge and the occurrence of testicular damage in animals submitted to one (G1) or three (G2) punctures. However, a higher percentage of tubular degeneration was associated to 18-gauge compared to 22-gauge fine needles in G3. In conclusion, multiple testicular needle aspiration can be safely conducted using fine needles. Large needles are recommended only for a single TNA, since multiple punctures may result in increased tubular degeneration and compromise testicular architecture and functionality.