Freshly collected boar spermatozoa are sensitive to a fast reduction in temperature because of lipid phase transition and phase separation processes. Temperature management during semen processing may determine the quality of stored samples. The aim of this study was to evaluate the influence of isothermic and hypothermic semen processing protocols on boar sperm quality under laboratory and field conditions. In the laboratory study, ejaculates (n = 12) were first diluted (1:1) with Beltsville Thawing Solution (BTS) at 32 °C, then processed either with isothermic (32 °C) or hypothermic (21 °C) BTS, stored at 17 °C, and assessed on days 1, 3, and 6. Temperature curves showed that 150 minutes after the first dilution, semen doses of both groups reached the same temperature. Two-step hypothermic processing resulted in lower sperm motility on days 1 and 6 (P < 0.05). Concomitantly, hypothermally processed samples contained less membrane intact sperm on days 3 and 6 (P < 0.05). Using AndroStar Plus extender instead of BTS reduced the negative effect of hypothermic processing. In the field study, 15 semen samples from each of 23 European artificial insemination studs were evaluated as part of an external quality control program. Semen quality based on motility, membrane integrity, mitochondrial activity, and a thermoresistance test was higher for stations using one-step isothermic dilutions (n = 7) compared with artificial insemination centers using two-step hypothermic protocols (n = 16). Both studies show that chilling injury associated with hypothermic dilution results in lower quality of stored boar semen compared with isothermic dilution and that the type of semen extender affects the outcomes.