Vertical connectivity between the surface channel and subsurface sediments may become increasingly important as climate change predictions forecast increased frequency of streambed drying in temporary streams in many regions. Streambed sediments can be a refuge for benthic macroinvertebrates during hydrological disturbances including streambed drying. A range of sedimentary characteristics may impede vertical connectivity and limit the potential of the subsurface to act as a refuge for aquatic macroinvertebrates during drying events. We directly observed the subsurface vertical movements of five lotic taxa, Nemoura cambrica (Plecoptera: Nemouridae); Hydropsyche siltalai (Trichoptera: Hydropsychidae); Heptagenia sulphurea (Ephemeroptera: Heptageniidae); Asellus aquaticus (Isopoda: Asellidae) and Gammarus pulex (Amphipoda: Gammaridae) in response to water level reduction using a variety transparent artificial microcosms containing different sediment sizes and interstitial pore space volumes. In addition, we examined the effect of varying fine sediment loads and reducing water level on the vertical movement of G. pulex in transparent mesocosms. The results from these novel experiments illustrate the importance of vertical connectivity and interstitial pathways to enhance faunal resistance and resilience in temporary streams and the need for their consideration in future restoration activities.