Do sex differences influence test habituation and internal data validity in neurocognitive testing? A blinded measurement error analysis

Reliable neurocognitive assessment requires sufficient habituation to ensure that test outcomes reflect stable cognitive performance rather than learning effects. This study examined the influence of repeated testing and sex differences on the reliability and internal validity of three widely used neurocognitive tasks: the Trail Making Test, Stroop Test (Word Read and Color Read), and CRT. One hundred healthy young adults (47 men, 53 women) completed all tasks twice daily over five consecutive days. Relative and absolute reliability, as well as agreement metrics were calculated to quantify systematic and random errors. Significant within- and between-days habituation effects were observed. Reliability varied substantially: the reaction tasks showed the highest stability, followed by Stroop tasks; the Trail-Making-Test B demonstrated the lowest reproducibility. Systematic improvements were most pronounced between sessions one and two and generally stabilized after two to four days of familiarization. Sex-specific analyses revealed consistent male superiority in choice reaction performance. Sex differences in habituation were task-dependent and primarily reflected differences in adaptation rate rather than the magnitude of improvement. Across sexes, sufficient task familiarization was essential to minimize systematic and random errors. Overall reliability metrics were similar across sexes. Maximal random errors were reported in the Trail-Making-Test, contradicting unhabituated test application to track longitudinal changes or establishing valid cross-sectional analyses.