AbstractThe endoplasmic reticulum (ER) is a single organelle in eukaryotic cells that extends throughout the cell and is involved in a large number of cellular functions. Using a combination of fixed and live cells (human MRC5 lung cells) in diffraction limited and super-resolved fluorescence microscopy (STORM) experiments, we determined that the average persistence length of the ER tubules was 3.03 ± 0.24 μm. Removing the branched network junctions from the analysis caused a slight increase in the average persistence length to 4.71 ± 0.14 μm, and provides the tubule’s persistence length with a moderate length scale dependence. The average radius of the tubules was 44.1 ± 3.2 nm. The bending rigidity of the ER tubule membranes was found to be 10.9 ± 1.2 kT (17.0 ± 1.3 kT without branch points). We investigated the dynamic behaviour of ER tubules in live cells, and found that the ER tubules behaved like semi-flexible fibres under tension. The majority of the ER tubules experienced equilibrium transverse fluctuations under tension, whereas a minority number of them had active super-diffusive motions driven by motor proteins. Cells thus actively modulate the dynamics of the ER in a well-defined manner, which is expected in turn to impact on its many functions.