Electrochemical aptamer-based (EAB) sensors are the first technology supporting high-frequency, real-time, in vivo molecular measurements that is independent of the chemical reactivity of its targets, rendering it easily generalizable. As is true for all biosensors, however, EAB sensor performance is affected by the measurement environment, potentially reducing accuracy when this environment deviates from the conditions under which the sensor was calibrated. Here, we address this question by measuring the extent to which physiological-scale environmental fluctuations reduce the accuracy of a representative set of EAB sensors and explore the means of correcting these effects. To do so, we first calibrated sensors against vancomycin, phenylalanine, and tryptophan under conditions that match the average ionic strength, cation composition, pH, and temperature of healthy human plasma. We then assessed their accuracy in samples for which the ionic composition, pH, and temperature were at the lower and upper ends of their physiological ranges. Doing so, we find that physiologically relevant fluctuations in ionic strength, cation composition, and pH do not significantly harm EAB sensor accuracy. Specifically, all 3 of our test-bed sensors achieve clinically significant mean relative accuracy (i.e., better than 20%) over the clinically or physiologically relevant concentration ranges of their target molecules. In contrast, physiologically plausible variations away from the temperature used for calibration induce more substantial errors. With knowledge of the temperature in hand, however, these errors are easily ameliorated. It thus appears that physiologically induced changes in the sensing environment are likely not a major impediment to clinical application of this in vivo molecular monitoring technology.