<p>The use of extracellular polymeric substances (EPS) stripped from sludge as a supplementary carbon source represents a novel method for enhancing nitrogen removal in low-carbon wastewater. This approach aims to leverage EPS while preserving the sludge metabolic activity. However, the effect of the EPS stripping degree on sludge metabolic activity remains poorly understood. In this study, EPS were progressively removed from activated sludge via ultrasonic centrifugation, and the resulting changes in sludge metabolic activity and settleability were systematically evaluated. The results demonstrate that key metabolic indicators, such as the specific oxygen uptake rate (SOUR), ammonia utilization rate (AUR), nitrogen utilization rate (NUR), and phosphorus release rate (PRR), initially increased and then decreased with increasing EPS removal. Optimal activity rates were observed at a 45% EPS stripping ratio, where SOUR, AUR, NUR, and PRR increased by 24%, 59%, 14%, and 9%, respectively, compared to the original sludge. Notably, the sensitivity to over-stripping varied: PRR inhibition commenced at 67% stripping, while NUR began to decline only after 86% stripping. In contrast, SOUR and AUR were enhanced across all tested stripping levels (up to 100%). This study is the first to establish a biological response relationship between the degree of EPS stripping and overall sludge metabolic activity, identifying 45% stripping as the optimum. It was also found that ultrasonic stripping reduced floc size and increased surface negative charge, thereby impairing sludge settleability. Consequently, practical application requires a balance between metabolic enhancement and sludge settleability.</p>