Background <p>Type-specific reference conditions are the anchor point of classifications of river ecological status. Usually based on the idea of a fixed and predictable status, they contrast with river dynamic hydrological trajectories. In this study, we focused on the impact of temporal streamflow variation and the related changes of lentic-lotic character of Mediterranean rivers on the definition of reference conditions for the assessment of their ecological status based on benthic macroinvertebrate community.</p> Results <p>Starting from 71-year long streamflow timeseries of a reference and a slightly altered river reach, located in the Sardinia Island, and using empirical relationships, we investigated the annual and monthly variability of the lentic-lotic character and of the biological metrics used to define the ecological status of Italian rivers (STAR_ICM index and its six sub-metrics) in different mesohabitats (pool and riffle). Then, we compared the result of the application of dynamic <i>vs.</i> fixed reference conditions for the STAR_ICMi calculation and ecological status classification. As expected, the lentic-lotic character of Sardinian rivers fluctuates continuously over time from lotic conditions in winter to extremely lentic conditions in summer, with significant variations from one year to another. All the biological metrics vary consequently over time, with a significant reduction in the summer months due to droughts/low flows and display a higher variability in pool than in riffle.</p> Conclusions <p>If dynamic instead of fixed reference conditions are used, quality classification shifts towards better ecological status, proving the potentially strong impact of ignoring the effects of natural habitat variability when assessing river ecological status.</p>