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The human microbiome is a dynamic community of bacteria, viruses, fungi, and other microorganisms. Both the composition of the microbiome (the microbes that are present and their relative abundances) and the temporal variability of the microbiome (the magnitude of changes in their composition across time, called volatility) has been associated with human health. However, the effect of unbalanced sampling intervals and differential read depth on the estimates of microbiome volatility has not been thoroughly assessed. Using four publicly available gut and vaginal microbiome time series, we subsampled the datasets to several sampling intervals and read depths and then compared additive, multiplicative, centered log ratio (CLR)-based, qualitative, and distance-based measures of microbiome volatility between the conditions. We find that longer sampling intervals are associated with larger quantitative measures of change (particularly for common taxa), but not with qualitative measures of change or distance-based volatility quantification. A lower sequencing read depth is associated with smaller multiplicative, CLR-based, and qualitative measures of change (particularly for less common taxa). Strategic subsampling may serve as a useful sensitivity analysis in unbalanced longitudinal studies investigating clinical associations with microbiome volatility.