Subsequence frequency measurement is a basic and essential problem in knowledge discovery in single sequences. Frequency based knowledge discovery in single sequences tends to be unreliable since different resulting sets may be obtained from a same sequence when different frequency metrics are adopted. In this chapter, we investigate subsequence frequency measurement and its impact on the reliability of knowledge discovery in single sequences. We analyse seven previous frequency metrics, identify their inherent inaccuracies, and explore their impacts on two kinds of knowledge discovered from single sequences, frequent episodes and episode rules. We further give three suggestions for frequency metrics and introduce a new frequency metric in order to improve the reliability. Empirical evaluation reveals the inaccuracies and verifies our findings.