Inefficient mixing in stirred vessels at low Reynolds numbers has been studied extensively over recent years. This paper investigates the mixing effects of square wave modulation protocol on isolated mixing regions (IMRs) in a stirred vessel using a Newtonian fluid. Glycerin was agitated using a standard six-bladed Rushton turbine inside an unbaffled tank. The impeller speed was modulated using a square wave protocol at two fixed amplitudes: A1 = 20 (Re{max} = 60 to Re{min} = 40) and A2 = 30 (Re{max} = 60 to Re{min} = 30) with different wave periods of 10 s and 40 s. The IMR structures was determined using a non-intrusive technique based on direct visualisation of an acid-base reaction with fluorescent green dye as a passive indicator. The volume of the IMR structures was determined using digital images taken during the mixing process. It was found that laminar mixing under Re < 100 could be enhanced by modulating the amplitude and also the wave period. It was also discovered that the IMR structures could be destroyed easily within Nt{m} (dimensionless mixing time) < 150 for upper envelope (UE) volume and Nt{m} < 463 for lower envelope (LE) volume using square wave protocol with shortest wave period and larger amplitude, thus producing a better mixing performance.