To date, there is only scarce evidence for a considerable association of subjective and objective stress measures, which might be attributable to method bias (e.g., confounding) and/or asynchrony of their temporal changes. To validate different subjective stress measures by a physiological measure of long-term stress (hair cortisol concentrations; HCC), 37 heterosexual couples (N = 74) completed a 12-week internet-based assessment protocol comprised of a weekly hassle scale (WHS; once per week), a perceived stress scale (PSS; once per month), and a chronic stress scale (TICS; once after three months). Partners provided vicarious stress ratings. When averaged across time, self-reported WHS significantly predicted HCC (r = 0.27), whereas the PSS and TICS did not (r < 0.22). Dynamic factor analysis (i.e., state-space modelling) confirmed that WHS was the most valid indicator of subjective stress, explaining up to 16% of the variance in HCC (r = 0.37) with a time lag of ~4 weeks. This temporally delayed effect of subjective stress is consistent with the presumed retrospective character of HCC, but also suggests that the majority of variance in hair cortisol is attributable to other causes than subjective stress such as individual disposition to display increased adrenocortical activity.