The mixing of slurry in an anaerobic digester (AD) is one of many key parameters, which have a significant effect on specific biogas yield (BY) and volatile solid (VS) removal rate. The determination of the optimum mixing regime in a digester is very complex as it depends on a large number of internal and external factors such as microbial community, the rheology of slurry, digester and impeller design, mixing intensity, and mixing intervals. The novelty of this study is the investigation of the optimum mixing regime in a lab-scale digester under semi-continuous mixing regimes by the continuous monitoring of the physicochemical properties of the digestate. In this study, a helical ribbon (HR) impeller was used for the agitation of the slurry operated at 67 rpm for 5 min under various agitation intervals (1 h, 2 h, 3 h, and 4 h). The results showed a 6–12% reduction in BY as the time between mixing operations increased. The highest BY was observed at a mixing frequency of 5 min/h, which produced a total of 54.1 L of biogas as compared to the mixing frequencies of 2 h, 3 h, and 4 h, where the BYs were recorded as 51.2 L, 49.8 L, and 47.3 L, respectively. Volatile fatty acids (VFAs) and FOS/TAC ratio were stabilized at 5–7 Gl−1 and 0.3–0.5, respectively. The appropriate mixing intensity was determined to obtain the highest biogas production, which could lead to lower power consumption for mixing operations.