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Abstract BackgroundPsychological stress is prevalent among reproductive‐aged men. Assessment of semen quality for epidemiological studies is challenging as data collection is expensive and cumbersome, and studies evaluating the effect of perceived stress on semen quality are inconsistent. ObjectiveTo examine the association between perceived stress and semen quality. Material and methodsWe analyzed baseline data on 644 men (1,159 semen samples) from two prospective preconception cohort studies during 2015–2021: 592 in Pregnancy Study Online (PRESTO) and 52 in SnartForaeldre.dk (SF). At study entry, men aged ≥21 years (PRESTO) and ≥18 years (SF) trying to conceive without fertility treatment completed a questionnaire on reproductive and medical history, socio‐demographics, lifestyle, and the 10‐item version of the Perceived Stress Scale (PSS; interquartile range [IQR] of scores: 0–40). After enrollment (median weeks: 2.1, IQR: 1.3–3.7), men were invited to perform in‐home semen testing, twice with 7–10 days between tests, using the Trak Male Fertility Testing System. Semen quality was characterized by semen volume, sperm concentration, and total sperm count. We fit generalized estimating equation linear regression models to estimate the percent difference in mean log‐transformed semen parameters by four PSS groups (<10, 10–14, 15–19, ≥20), adjusting for potential confounders. ResultsThe median PSS score and IQR was 15 (10–19), and 136 men (21.1%) had a PSS score ≥20. Comparing men with PSS scores ≥20 with <10, the adjusted percent difference was −2.7 (95% CI: −9.8; 5.0) for semen volume, 6.8 (95% CI: ‐10.9; 28.1) for sperm concentration, and 4.3 (95% CI: −13.8; 26.2) for total sperm count. ConclusionOur findings indicate that perceived stress is not materially associated with semen volume, sperm concentration, or total sperm count.