Ecologists and zoologists have worked for centuries to understand the spreading, extent, and regulation of biological rhythms among eukaryotes living in subterranean ecosystems. Subterranean habitats generally lack diel variation in environmental conditions. Investigating whether the long-standing hypothesis that biological clocks went lost in subterranean-dwelling species can thus have broad implications in assessing evolutionary and ecological pathways allowing colonization of new and physiologically challenging environments. However, chronobiological research in cave species remains hindered by methodological, ethical, and logistical constraints, particularly for field-based studies. Here, we propose a flexible, non-invasive framework to assess temporal variation in the activity of subterranean animals using detection probability as a proxy for biological rhythms. Our approach is based on Bayesian occupancy and abundance models, which can incorporate quantitative (count), qualitative (presence/absence), or semi-quantitative (abundance classes) data derived from repeated surveys. This framework is adaptable to a variety of taxa, monitoring strategies, and environmental conditions. It is particularly well-suited for sensitive ecosystems, as it minimizes disturbance and allows for standardized, repeatable analysis of activity patterns. We illustrate the utility of the framework through three case studies (Proteus anguinus Laurenti 1768, Niphargus sp. Schiødte 1849 and Spelaeomysis bottazzii Caroli 1924) encompassing different data types and showing its potential to detect rhythmicity even in stable subterranean environments. Our method provides a novel and accessible tool for exploring biological rhythms in underground environments. By facilitating robust in situ assessments of temporal activity, it can contribute to a deeper understanding of rhythm evolution in extreme habitats and support the conservation of highly specialized, often vulnerable, subterranean fauna.
