The microtubule (MT)–kinesin system is a promising candidate for constructing artificial biomachines. The active self-organisation (AcSO) method has been developed to integrate MT filaments into highly organised assembled structures. The creation of ring-shaped MT assemblies is one of the outcomes of the organisation process and holds prospects for use in future nano-technological applications. However, making use of ring-shaped MT assemblies in practical applications requires further control of the size of these assemblies, which has not yet been addressed. In this work, we demonstrated AcSO of MTs in a stepwise manner inside an inert atmosphere. We show that in an inert atmosphere, AcSO could be performed several times (at least nine times), and as a result, this method successfully increased the thickness of ring-shaped MT assemblies.