Scanning electrochemical microscopy (SECM) is being used increasingly to monitor electrochemical processes at the interface of living cells and electrodes. This allows the detection and quantification of biomarkers that further the understanding of various diseases. Rapid SECM experiments are often carried out without monitoring the analyte solution temperature or are performed at room temperature. The reported research demonstrates that temperature control is crucial during SECM imaging of living cells to obtain reliable data. In this study, a SECM-integrated thermostatic ring on the sample stage enabled imaging of living biological cells in a constant height mode at various temperatures. Two-dimensional line scans were conducted while scanning single Adenocarcinoma Cervical cancer (HeLa) cells. Numerical modeling was carried out to evaluate the effect of the temperature on the electrochemical current response of living cells to compare the apparent heterogeneous rate constant (k0), representing cellular reaction kinetics. This study reveals that even slight temperature variations of approximately 2 °C affect the reaction kinetics of single living cells, altering the measured current during SECM.