Human blood cells may offer a minimally invasive strategy to study systemic alterations of mitochondrial function. Here we tested the reliability of a protocol designed to study mitochondrial respiratory control in human platelets (PLTs) in field studies, using high-resolution respirometry (HRR). Several factors may trigger PLT aggregation during the assay, altering the homogeneity of the cell suspension and distorting the number of cells added to the two chambers (A, B) of the Oroboros Oxygraph-2k (O2k). Thus, inter-chamber variability (∆ab) was calculated by normalizing oxygen consumption to chamber volume (JO2) or to a specific respiratory control state (flux control ratio, FCR) as a reliable parameter of experimental quality. The method’s reliability was tested by comparing the ∆ab of laboratory-performed experiments (LAB, N = 9) to those of an ultramarathon field study (three sampling time-points: before competition (PRE, N = 7), immediately after (POST, N = 10) and 24 h after competition (REC; N = 10)). Our results show that ∆ab JO2 changed PRE-POST, but also for LAB-POST and LAB-REC, while all ∆ab FCR remained unchanged. Thus, we conclude that our method is reliable for assessing PLT mitochondrial function in LAB and field studies and after systemic stress conditions.