Pullout strength is an important indicator of the performance and longevity of pedicle screws and can be heavily influenced by the screw design, the insertion technique and the quality of surrounding bone. The purpose of this study was to investigate the pullout strength of three different pedicle screws inserted using three different strategies and with two different loading conditions. Three pedicle screws with different thread designs (single-lead-thread (SLT) screw, dual-lead-thread (DLT) screw and mixed-single-lead-thread (MSLT) screw) were inserted into a pre-drilled rigid polyurethane foam block using three strategies: (A) screw inserted to a depth of 33.5 mm; (B) screw inserted to a depth of 33.5 mm and then reversed by 3.5 mm to simulate an adjustment of the tulip height of the pedicle screw and (C) screw inserted to a depth of 30 mm. After insertion, each screw type was set up with and without a cyclic load being applied to the screw head prior to the pullout test. To ensure that the normality assumption is met, we applied the Shapiro–Wilk test to all datasets before conducting the non-parametric statistical test (Kruskal–Wallis test combined with pairwise Mann–Whitney-U tests). All screw types inserted using strategy A had a significantly greater pullout strength than those inserted using strategies B and C, regardless of if the screw was pre-loaded with a cyclic load prior to testing. Without the use of the cyclic pre-load, the MSLT screw had a greater pullout strength than the SLT and DLT screws for all three insertion strategies. However, the fixation strength of all screws was reduced when pre-loaded before testing, with the MSLT screw inserted using strategy B producing a significantly lower pullout strength than all other groups (p< 0.05). In contrast, the MSLT screw using insertion strategies A and C had a greater pullout strength than the SLT and DLT screws both with and without pre-loading. In conclusion, the MSLT pedicle screw exhibited the greatest pullout strength of the screws tested under all insertion strategies and loading conditions, except for insertion strategy B with a cyclic pre-load. While all screw types showed a reduced pullout strength when using insertion strategy B (screw-out depth adjustment), the MSLT screw had the largest reduction in pullout strength when using a pre-load before testing. Based on these findings, during the initial screw insertion, it is recommended to not fully insert the screw thread into the bone and to leave a retention length for depth adjustment to avoid the need for screw-out adjustment, as with insertion strategy B.