Objectives: The aims of this study are to investigate the effects of diplophonia on jitter and shimmer and to identify measurement limitations with regard to material selection and clinical interpretation. Materials and Methods: Four hundred and ninety-eight audio samples of sustained phonations were analyzed. The audio samples were assessed for the grade of hoarseness and the presence of diplophonia. Jitter and shimmer were reported with regard to perceptual ratings. We investigated cycle marker positions exemplarily and qualitatively to understand their implications for perturbation measurements. Results: Medians of jitter and shimmer were higher for diplophonic voices than for nondiplophonic voices with equal grades of hoarseness. The variance of jitter for moderately dysphonic voices was larger than the variance observed in a corpus from which diplophonic samples had been discarded. The positions of cycle markers in diplophonic voices did not match the positions of the pulses, indicating that the validity of jitter and shimmer values for these voices were questionable. Conclusion: Diplophonia biases the reporting of dysphonia severity via perturbation measures, and their validity is questionable for these voices. In addition, diplophonia is an influential source of variance in jitter measurements. Thus, diplophonic fragments of voice samples should be excluded prior to perturbation analysis.