While MRI and CT are the gold standards for assessments of splenic size in clinical settings, ultrasonography is particularly suited due to its portability, cost efficiency and easy utilization. However, ultrasonography is associated with subjective assessment, potentially resulting in increased variation. We used a test–retest design aiming to determine the reliability of splenic measurements assessed by ultrasonography during apnea. In addition, we compared reliability between different equations for volume calculations: Koga, Prolate ellipsoid and Pilström. Twelve healthy participants (6 women) performed two tests separated by 15 min, comprising a maximal voluntary apnea in a seated position. Splenic dimensions were measured via ultrasonography for 5 min before and immediately following apnea. Resting splenic volume displayed high test–retest reliability between tests (Pilström: 157 ± 39 mL vs 156 ± 34 mL, p = .651, ICC = .970, p < .001, CV = 2.98 ± 0.1%; Prolate ellipsoid: 154 ± 37 mL vs 144 ± 43 mL, p = .122, ICC = .942, p < .001, CV = 5.47 ± 0.3%; Koga: 142 ± 37 mL vs 140 ± 59 mL, p = .845, ICC = .859, p < .001, CV = 9.72 ± 1.4%). Apnea-induced volumes displayed similar reliability (127 ± 29 mL vs 129 ± 28 mL, p = .359, ICC = .967, p < .001, CV = 3.14 ± 3.1%). Reliability was also high between equations (Pilström vs Prolate ellipsoid: ICC = .818, p < .001, CV = 7.33 ± 0.3%, bias =  − 3.1 mL, LoA =  − 46.9 to 40.7 mL; Pilström vs Koga: ICC = .618, p < .01, CV = 11.83 ± 1.1%, bias =  − 14.8 mL, LoA =  − 76.9 to 47.3 mL). We conclude that splenic ultrasonographic measurements have practical applications during laboratory and field-based research as a reliable method detecting splenic volume change consistently between repeated tests. The Pilström equation displayed similar reliability compared to the prolate ellipsoid formula and slightly higher compared to the Koga formula and may be particularly useful to account for individual differences in splenic dimensions.