Background <p>Current approaches to prescribing exercise intensity might not standardize physiological strain across individuals, potentially affecting training outcomes. Whether exercise guidelines applying a 1:2 duration ratio between moderate (MOD) and vigorous (VIG) activities equalize physiological strain is unclear. This exploratory study compared physiological strain and its variance across two intensities and three prescription methods.</p> Methods <p>Thirteen habitually active males performed an incremental treadmill test as well as 40-min MOD and 20-min VIG sessions prescribed by: (1) absolute metabolic equivalents (ABS), (2) relative maximal oxygen uptake (VO_2max) (REL), and (3) lactate thresholds (LT). Physiological strain was estimated using excess post-exercise oxygen consumption, individualized training impulse, post-exercise heart rate variability (HRV), blood lactate, and session rating of perceived exertion (sRPE).</p> Results <p>Except for sRPE, estimated physiological strain was greater during VIG than MOD (<i>p</i> &lt; 0.05) across all prescription methods. A general linear mixed model estimated that equivalent strain for most outcomes would have required MOD &gt; 55% and VIG &lt; 72% of VO_2max. According to modified Levene’s test, variance in physiological strain did not differ significantly between prescription methods in MOD. In blood lactate, the variance was higher in VIG_REL than VIG_LT (<i>p</i> = 0.025), and HRV variance was greater in VIG_ABS than VIG_LT (<i>p</i> = 0.041).</p> Conclusion <p>The 1:2 duration ratio underestimated physiological strain of VIG sessions regardless of prescription method. Based on the observed differences in variance, the appropriate prescription method may become increasingly important at higher intensities. However, analyses indicated substantial uncertainty in the magnitude of variance, highlighting the need for large-scale studies on the topic.</p>