This paper concerns non-overlapping codes, block codes motivated by synchronisation and DNA-based storage applications. Most existing constructions of these codes do not account for the restrictions posed by the physical properties of communication channels. If undesired sequences are not avoided, the system using the encoding may start behaving incorrectly. Hence, we aim to characterise all non-overlapping codes satisfying two additional constraints. For the first constraint, where approximately half of the letters in each word are positive, we derive necessary and sufficient conditions for the code's non-expandability and improve known bounds on its maximum size. We also determine exact values for the maximum sizes of polarity-balanced non-overlapping codes having small block and alphabet sizes. For the other constraint, where long sequences of consecutive equal symbols lead to undesired behaviour, we derive bounds and constructions of constrained non-overlapping codes. Moreover, we provide constructions of non-overlapping codes that satisfy both constraints and analyse the sizes of the obtained codes.