Background: Sperm capacitation remains one of the major challenges limiting the development of reliable conventional in vitro fertilisation (IVF) in the horse. This study evaluated the effects of medium, incubation time, and capacitating supplements, including sodium metabisulfite (SMB), on stallion sperm function. SMB is a chemical stabiliser commonly used to maintain the stability of the penicillamine-hypotaurine-epinephrine (PHE) capacitation system. Methods: Semen samples were collected from four pony stallions, purified by single-layer colloid centrifugation, and incubated for 22 h at 37°C under 5% CO2. Sperm were incubated in either Biggers, Whitten and Whittingham (BWW) or Tyrode's Albumin Lactate Pyruvate (TALP) medium supplemented with PHE, PHE+SMB, SMB, or no supplement (control). Sperm motility was evaluated using computer-assisted sperm analysis, while viability, acrosome status, and protein tyrosine phosphorylation were assessed by flow cytometry. Data were analysed using linear mixed-effects models. Results: BWW consistently maintained higher total motility, viability, and total live acrosome-reacted spermatozoa than TALP, with differences becoming evident after 3 h. At 9 h, total motility in BWW was more than twice that observed in TALP (72.1 ± 3.3% vs. 28.8 ± 2.7%). Significant Media × Time and Stallion × Time interactions were detected for most motility parameters (p<0.05), whereas treatment had no significant effect on sperm motility, viability, or acrosome reaction (p>0.05). Nevertheless, SMB consistently showed the highest numerical values of viable tyrosine-phosphorylated and acrosome-reacted spermatozoa. Temporal changes in the hyperactivation-associated kinematic parameters (VCL, ALH, and LIN), together with increased protein tyrosine phosphorylation, were consistent with sperm capacitation during incubation. Conclusion: BWW provides a more favourable environment than TALP for supporting stallion sperm capacitation and viability. SMB may contribute to promoting sperm capacitation. The results also identify 3-9 h as the optimal incubation window for conventional equine IVF. Marked stallion-to-stallion variation further highlights the importance of stallion-specific optimisation of IVF protocols.