In multitasking environment, the response time is unpredictable because the execution speed of measurement and control tasks is uncertain. In order to solve this problem, first, a response time model is put forward based on the virtual instrument architecture and the real-time working conditions. Next, real-time measuring indicators are exactly computed via the timing measurement at a microsecond level. Then, the main approaches to real- time performance optimization are proposed and experimentally verified based on the derivation of data-processing delay formula. Finally, an estimation formula for the highest data acquisition frequency is presented based on the analysis of experimental results, which enables virtual instruments to maintain stable response time under the system load as high as that with 80% CPU usage, and thus realizes the constrained optimization of real-time performance.